WO2008057074A1 - Procédé pour une chromatographie à écoulement - Google Patents

Procédé pour une chromatographie à écoulement Download PDF

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Publication number
WO2008057074A1
WO2008057074A1 PCT/US2006/043135 US2006043135W WO2008057074A1 WO 2008057074 A1 WO2008057074 A1 WO 2008057074A1 US 2006043135 W US2006043135 W US 2006043135W WO 2008057074 A1 WO2008057074 A1 WO 2008057074A1
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WIPO (PCT)
Prior art keywords
media
beads
agarose
column
protein
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PCT/US2006/043135
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English (en)
Inventor
Chen Wang
Kwok-Shun Cheng
Senthilkumar Ramaswamy
Nanying Bian
Brian Gagnon
Joaquin A. Umana
Dennis Aquino
Neil Soice
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Millipore Corporation
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Priority to PCT/US2006/043135 priority Critical patent/WO2008057074A1/fr
Publication of WO2008057074A1 publication Critical patent/WO2008057074A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3291—Characterised by the shape of the carrier, the coating or the obtained coated product
    • B01J20/3293—Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/26—Cation exchangers for chromatographic processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J41/00—Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/20—Anion exchangers for chromatographic processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography

Definitions

  • the present invention relates to a method of purification using chromatography media and equipment. More particularly it relates to a method of flow-through chromatography using cored beads.
  • the chromatograph steps is run in a flow-through manner.
  • this is the final polishing chromatography step which involves flowing the Mab solution through an anion exchange media under conditions where the Mab does not bind to the media but negatively charged impurities will bind, such as DNA, viruses and host cell proteins (HCP or CHOP).
  • HCP host cell proteins
  • chromatography beads provide excellent impurity removal at bed heights around 20cm.
  • the column is often sized (in diameter) to provide permeability, resulting in a much oversized column in terms of its total protein capacity (20 Kg BSA capacity for typical 200L column).
  • the bead columns are oversized because the packed bed height is limited in available flow rates due to bead compressibility. Bead compression causes a reduction in the ability of the bead to bind the desired impurities (capacity) and can if severe enough lead to a permanent reduction in capacity if the beads become irreversibly compressed. Therefore to gain the desired volume one expands the bed laterally, avoiding bead compression.
  • Membrane chromatography devices overcome this permeability challenge by achieving excellent impurity removal at very low bed heights ( ⁇ 1-2cm). However, for membrane devices this low bed height results in much lower protein capacities (-300 g BSA for 5L device). This reduction in protein capacity greatly reduces the "safety factor" for protein removal that exists with bead-based columns. It is important to note that a small scale, such as lab and early pilot scale, the differences in protein capacity between bead columns and available membrane devices is small. At small scale, it is much easier to size a membrane device that can compete in protein capacity with a small scale bead column.
  • chromatography beads such as Sepharose® Fast Flow media from GE
  • Sepharose® Fast Flow media from GE
  • a major weakness of this approach is the limited mechanical properties of the agarose bead media.
  • a 20cm bed height of beads can be run at a flow rate up to 300 cm/hr.
  • This permeability limitation results in the use of a larger diameter column (because it is not possible to increase the bed height).
  • This larger diameter column results in a huge over capacity/safety factor for proteins and a large column footprint which is a problem for space limited facilities. Therefore, a media that can offer high permeability, good protein safety factor and ease of scale-up is needed for polishing applications.
  • Chromatography beads constructed of polysaccharides were initially developed in the 1960s. These materials have been commercially successful because of their high protein capacity, low non-specific binding and chemical stability. One deficiency of these materials is they are inherently compressible. Extensive work has been conducted using chemical modification to reduce this compressibility through crosslinking of the polysaccharides.
  • Filled polysaccharide beads are well established for uses including expanded bed or fluidized bead chromatography. These materials are made by adding one or more non- porous spheres to the polysaccharide (typically agarose) during bead formation. In this manner, a bead with one or more non-porous particles encapsulated inside the polysaccharide material can be formed. In some cases, the non-porous particle can be centered such that a pellicular chromatography bead is formed. The non-porous particle(s) serves no function in the protein capacity or separation properties of the bead. The particle(s) typically acts only to modify the density of bead such that the material can be used for non- packed bed applications.
  • Anion exchange beads such as Sepharose® Fast Flow Q media can be packed in columns up to 20cm bed height.
  • the volume of media is typically 1 liter/50g of Mab.
  • a 10,000 Liter batch of a titer of 1 g/L a 200 liter column would be needed for the polishing step.
  • the feed After the cation exchange step, the feed would be 10 g/L at a volume of 1000 liters.
  • the time to process this volume at 76 cm/hr (published column run condition) would be about 79 minutes.
  • This process step usually has >5 Log (or 99.999%) removal of virus and can reduce host cell protein (HCP or CHOP) to ⁇ 2ng/mg antibody.
  • HCP or CHOP host cell protein
  • This device has a protein capacity about 50 g/L and can process feed at up to 50 L/minute. Therefore, to purify 10 Kg of MAb in 1000L it would take about 20 minutes.
  • the drawback to membrane chromatography is the complexity and cost of making larger volume devices. These limitations force membrane devices to handle more feed per liter of media. This additional challenge can sometimes reduce the media's performance in the flow-through polishing step.
  • the protein capacity is about 20g/L (Safety factor of 10Ox). Under the conditions described here, CHOP is reduced to ⁇ 2 ng/mg Mab . The maximum flow rate is 4 L/min. This results in a processing time of 250 minutes for a 10 Kg Mab batch. Furthermore, at feed loadings of 5,000 g/ liter of membrane the virus removal was less than 1 Log.
  • Membranes provide an alternative to beads that allows for the used of smaller media volumes and higher flow rates.
  • the invention is to a method for purifying proteins using a bed of "cored beads" in a flow-through or polishing chromatography mode.
  • a pellicular agarose/non-porous "cored bead” media provides one with media that have much improved permeability and mechanical properties over existing beads, while maintaining an increased protein "safety factor” and ease of scale-up over existing membrane devices.
  • Cored agarose media use rigid internal beads (in this case beads of non-porous, synthetic polymer, metal or glass) which form incompressible and highly permeable packed beds especially when the agarose coating is relatively thin (e.g.5-15 microns on a greater than 50 micron diameter core).
  • Figure 1 shows a comparison of the media of the prior art with the media according to the present invention.
  • Figure 2 shows a graph of the dynamic capacity of the present invention.
  • Figure 3 shows a graph of the increased protein capacity of the present invention.
  • Figure 4 shows the viral removal capability of the present invention.
  • Figure 5 shows the host cell protein assay results for the present invention.
  • the invention is a method/application for purifying proteins using a bed of "cored beads” in a flow-through or polishing chromatography mode.
  • "cored bead” media can provide much improved permeability and mechanical properties over existing beads, while maintaining an increased protein "safety factor” and ease of scale-up over existing membrane devices.
  • Cored agarose media 2 using rigid internal beads 4 in this case beads of non-porous, synthetic polymer, metal or glass
  • Figure 1 can form incompressible and highly permeable packed beds especially when the agarose coating 6 is relatively thin (e.g.5-15 microns on a greater than 50 micron diameter core).
  • homogeneous agarose media 8 in Figure 1 are quite compressible even when highly crosslinked.
  • the decreased media gel volume of the cored ' media will result in enhanced permeability and a lower protein capacity (especially in terms of g protein/ liter of media) than homogeneous media.
  • the protein capacity for beads is usually in great excess during finial polishing step, an improved overall performance can be achieved by trading off some of the capacities (or gel volume) for permeability and/or rigidity. For instance, with improved permeability and rigidity, a longer column can be packed and operated at a faster flow rate, thus reducing the total processing time for a given batch size.
  • these materials can be used in applications where highly permeable beds are desired because one can tune the capacity (changing the coating thickness, resulting in different adsorbant volume fraction in the bead), permeability (core and bead sizes) and bed rigidity (gel thickness and agarose concentration) to maximize throughput or minimize processing time.
  • the desired product does not bind to the media and only trace impurities such as virus, DNA and CHOP are absorbed.
  • concentration of impurities is usually small ( ⁇ 100 ng/mg of Mab) which means the total concentration of impurity for a 10 Kg Mab batch would be ⁇ 1 g. Therefore, having a protein capacity of ⁇ 20Kg (in a typical Sepharose®-Q Fast Flow column) is an enormous safety factor (20,00Ox).
  • a final advantage of an incompressible cored media is the possibility of prepacking the bead media. Due to the incompressible nature of the media, it can be easily prepacked using mechanical means in which the packing end point can be easily observed by the large increase in resistance. The column could be shipped to the customer pre-wet with a sterilizing solution allowing for essentially disposible chromatography.
  • a cored bead can be made by the process as taught in US 5,935,442 which relates to density controlled agarose beads for expanded bed chromatography.
  • the cored beads are made by the process of our co-pending application entitled “Method and Apparatus for Making Porous Agarose Beads " filed this same day the teachings of which is incorporated herein by reference.
  • this process one adds agarose to an aqueous solution and emulsifier and heats it to a temperature at or above the melting point of the agarose (typically from about 80 0 C to 12O 0 C).
  • the cores are then added to the agarose solution.
  • the heated solution is then added to a heated hydrophobic liquid, such as mineral oil or other oils and polymers, at a temperature at about or above the melting point of the agarose to form an emulsion.
  • a heated hydrophobic liquid such as mineral oil or other oils and polymers
  • the liquid is agitated to increase the emulsion formation.
  • the emulsion is then flowed through one or more static mixers to create beads having a core and an agarose coating.
  • Suitable static mixers include for example Kenics static mixers (Model KMR-SAN-12, 0.5 inch (12.7mm) diameter, 12 element mixer) and a Ross ISG static mixer with a 0.5 inch (12.7mm) diameter and 10 elements.
  • Other static mixers are commercially available and would be useful in the present invention as well.
  • the beads are flowed into a cooled bath of hydrophobic liquid (typically from about 1 0 C to about 7O 0 C, preferably from about 1 0 C to about 4O 0 C and most preferably about 5 0 C) to cause the agarose to gel on the core.
  • hydrophobic liquid typically from about 1 0 C to about 7O 0 C, preferably from about 1 0 C to about 4O 0 C and most preferably about 5 0 C
  • the beads are then recovered, washed one or more times with water and are then crosslinked and have a functionality (preferably anionic or cationic) bonded to its surfaces.
  • single core yield can be as high as 80%.
  • the single core yield is at least 50%. While water is the preferred solvent for the agarose, a minor amount, up to 20% by weight of the aqueous solution, of one or more co- solvents may be added to improve the solubility of the agarose. Examples of suitable co- solvents are dimethylacetamide and/or dimethylsulfoxide. Others are known to those skilled in the art.
  • the cores of the coated agarose of the present invention can be made of any material that is useful in chromatography.
  • the core may be a crossed linked agarose bead (whether made by the present process or any other process), a plastic, metal, glass or ceramic.
  • the core is selected from a material that is doesn't melt at the temperatures used in the present process and which is self-supportive.
  • Suitable materials include but are not limited to plastics such as polystyrene, polyethylene, polypropylene, blends of polyethylene and polypropylene, multilayered polyethylene/polypropylene beads, acrylics, polysulfones, polyethersulfones, PVDF or PTFE; glass such as borosilicate glass, alkali resistant glass and controlled pore glass, metals such as stainless steel, nickel, titanium, palladium and cobalt or various iron, iron containing or other magnetized metals alloys and blends; and ceramics, such as silicate materials, zirconia and various ceramic blends.
  • plastics such as polystyrene, polyethylene, polypropylene, blends of polyethylene and polypropylene, multilayered polyethylene/polypropylene beads, acrylics, polysulfones, polyethersulfones, PVDF or PTFE
  • glass such as borosilicate glass, alkali resistant glass and controlled pore glass, metals such as stainless steel, nickel,
  • the cores are preferably of a generally spherical or irregular particulate shape. Their diameter depends upon the size of bead one desires but preferably are from about 50 microns to about 150 microns in diameter, more preferably from about 70 to about 120 microns.
  • the agarose coating formed on the bead should be of sufficient thickness so as to provide the necessary physical strength and the depth and width of porosity required for a given application. It should be thin enough to provide for fast permeability of the impurities into its porous structure.
  • the coating is from about 3 microns to about 15 microns thick. More preferably the agarose coating is from about 4 to about 10 microns thick. Most preferably, it is from about 4 to about 7 microns thick.
  • the pore size of the gel coating is primarily determined by agarose concentration and the extent of chemical crosslinking. For flow through application, relatively small protein impurities penetrate into the gel matrix while Mab partition into the gel should be minimized. Thus a gel pore size that is similar to or even tighter than a typical gel for Mab binding would be preferable.
  • the gel concentration ranges from 4% to 12%, and more preferably, from 6% to 8%.
  • the resulting gel pore radius is in the range of 2 to 40 nm, and more preferably, from 5 to 20 nm.
  • the porosity of the gel is dependent upon the agarose concentration.
  • the porosity of the coating is in the range of 80 to 96% based on the gel volume, and preferably from 85% to 94%.
  • the corresponding bed porosity is in the range of 40 to 75% and preferably from 55 to 70%.
  • various additives can be used to enhance production or add a property to the beads.
  • One class of additives comprises volatile organics, miscible with the solution.
  • examples are monohydric alcohols such as methanol, ethanol, and propanols. These can be used up to concentrations that give a slightly cloudy solution. Higher amounts of these alcohols can cause precipitation of the agarose.
  • Miscible ketones such as acetone can also be used, but care must be used as the solubility of agarose is less in ketone-water mixtures. Any mixture of two or more of these materials is also contemplated.
  • a further class of additives comprises non-volatile miscible organics.
  • Polyethylene glycols of low molecular weight are also examples of materials that
  • Another class of additives comprises water-soluble polymers, which include by way of examples, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycols, dextrans, and water-soluble polyacylamides, including substituted polyacylamides, such as polydimethylacrylamide.
  • These polymeric additives can be used as blends with the agarose in the initial dissolution step, or they can be dissolved in the solution after the addition and dissolution of the agarose. Care must be taken not to add an excessive amount of polymer, as coagulation of the solution may occur. Ratios of polymer to agarose of from about 0.1 to 10 are possible.
  • Preferred polymers are polyvinyl alcohol, dextrans and polyacrylamides.
  • salts include by way of examples, sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, sodium iodide and sodium phosphate.
  • the addition of salt can change the solubility, gel point and viscosity of the dissolved agarose solution. Any mixture of two or more of these materials is also contemplated.
  • the agarose may then be crosslinked if desired by any of the chemistries commonly used in the industry to crosslink materials containing multiple hydroxyl groups, such as polysaccharide beads, these chemistries being as non-limiting examples, epichlorohydrin or other multifunctional epoxy compounds, various bromyl chemistries or other multifunctional halides; formaldehyde, gluteraldehyde and other multifunctional aldehydes, bis(2-hydroxy ethyl)sulfone, dimethyldichloro-silane, dimethylolurea, dimethyiol ethylene urea, diisocyanates or polyisocyanates and the like.
  • chemistries commonly used in the industry to crosslink materials containing multiple hydroxyl groups
  • these chemistries being as non-limiting examples, epichlorohydrin or other multifunctional epoxy compounds, various bromyl chemistries or other multifunctional halides; formaldehyde, gluteraldehyde and other multifunctional al
  • the desired functionality is applied to the agarose coating on the beads to render the agarose cationic or anionic, as is well-known in the art of media formation.
  • These groups may be added after the cored agarose bead has been formed and crosslinked or they may be added to the initial solution and the composition of the initial solution is modified accordingly, such as pH being lowered or raised, so that the reaction to link the functional groups to the agarose occurs concurrently with the crosslinking reaction.
  • these functionalities are commonly applied in two ways. First these functionalities can be applied with the addition of an electrophile containing molecule which possesses the desired functional group or a precursor thereof.
  • the hydroxyl containing base matrix is activated with sodium hydroxide which allows for efficient reaction of the base matrix with the aforementioned electrophile.
  • Non-limiting examples include: bromopropane sulfonic acid, propane sultone, allyl glycidyl ether, allyl bromine, glycidyl trimethylammonium chloride, butanediol diglycidyl ether, sodium chloroacetate.
  • a nucleophilic group such as an amine or thiol
  • an electrophilic group including the following non-limiting samples, cyanogen bromide, activated carboxylic acids, aldehydes, esters, epoxides such as butanediol diglycidyl ether, epichlorohydrin, allyl bromide and allyl glycidyl ether, followed by reaction of the activated base matrix with the appropriate nucleophilic molecule containing the functionality of choice.
  • These nucleophiles can be small molecules such as aminopropane sulfonic acid or larger entities such as polyethyleneimine or proteins and peptides.
  • a suitable method of using the cored beads in the present process is to pack a suitably sized column with a slurry of cored beads described herein.
  • the beads may have a functionality selected from the group consisting of anions and cations.
  • the bed of beads is compacted to form an uniformly packed bed within the column as is well-known in the art.
  • a feedstock containing the protein of interest, such as a monclonal antiboby (Mab), with or more impurities is fed to the column and passed through the column so that the impurities are absorbed to the beads and the desired protein passes through.
  • the column is then washed with one or more eluant washes to dislodge the impurities which are then sent to waste.
  • the column is then regenerated and the media cleaned and sterilized for its next use.
  • the beads are relatively inexpense, they may simply be sent to waste after a single use.
  • the feedstock that enters the flow through polishing media usually contains less than 10 7 /ml of virus, ⁇ 1 ⁇ g/ml of DNA, ⁇ 100 EU/ml of endotoxin, and ⁇ 100 ng/ml of HCP, and the media needs to reduce these impurities to acceptable levels to meet the final product specifications.
  • the permeability of the chromatography media can be defined as the product of flow velocity and bed length per units of pressure drop.
  • Typical homogeneous agarose media with a particle size distribution similar to Q-Sepharose® Fast Flow media has permeability in the range of 400 to 1000 cm 2 /hr/psi.
  • the cored beads media can provide 1.5 to 4-fold higher permeability (from about 600 to about 4000 cm 2 /hr/psi) at similar or slightly larger average bead size.
  • the conventional agarose beads are usually packed to 15 to 25 cm in length. Since the cored beads have enhanced permeability, the column may be packed from 15 to 60 cm without causing significant bed compression. Depending on the bed length, the maximal operating flow rate may vary. For a typical bed length (i.e., 20 cm) and maximal pressure drop of 2 bar, the cored beads can be flow from 50 cm/hr up to 1000 cm/hr for beads of smaller size or 2800 cm/hr for beads of larger size.
  • the binding efficiency of the impurities can be tuned by varying the bead size and surface chemistry. Smaller bead size and stronger binding strength enhance the binding kinetics, especially for virus and HCP (or CHOP). Good virus and HCP removal can be obtained by cored beads with diameter of 50 to 120 urn and charge density similar to or higher than that of Q-Sepharose® FF media.
  • the binding strength may be also enhanced by tuning the gel pore size and surface hydrophobicity.
  • the cored beads can provide a LRV of 3 to 7 for virus, and 1 to 2 for HCP. Endotoxin and DNA removal should be of no problem by the cored anion exchange media due to their excessive negative charges.
  • Coated beads formed as described above are crosslinked and then subjected to one or more additional coating processes by mixing the crosslinked coated beads with a heated, dissolved agarose in an aqueous solution as described above, flowing the coated beads and agarose into a first heated hydrophobic liquid containing an emulsifierto create an emulsion, flowing the emulsion through a static mixed as described above into a second liquid cooled to a temperature below the melting point of the agarose to create the additional coating layer on each bead and then recovering the beads for further processing.
  • additional coats are desired the agarose on the bead should be crosslinked before applying the additional coating to prevent its dissolution in the heated solution or first bath.
  • Example 1 Anion Exchange Bead (80 ⁇ m) with 70 ⁇ m borosilicate core and agarose 5 ⁇ m coating.
  • Agarose coated borosilicate beads were made using a process described in our co- pending application entitled “Method and Apparatus for Making Porous Agarose Beads" filed this same day.
  • the agarose coated beads (80 ⁇ m diameter, 5 ⁇ m coating, 6% agarose, 50 mL volume) were crosslinked using epichlorohydrin according to the well-known method of the prior art.
  • Anion exchange groups (quaternary amine groups) were added with the following method: 5OmL of beads were added to 50 mL of 75% glycidyl trimethylammonium chloride (GTMAC). To this mixture, 1.67 mL of 50% wt sodium hydroxide was added. The reaction was stirred at room temperature overnight. The beads were than filtered and washed with three 200 mL volumes of Milli-Q® water. The beads were stored in 20% ethanol/water solutions.
  • GTMAC glycidyl trimethylammonium chloride
  • Example 2 Anion Exchange Bead (100-116 ⁇ m) with 90-106 ⁇ m borosilicate core and 5 ⁇ m coating.
  • Example 2 was made exactly as Example 1 , except the starting bead consisted of a 5 ⁇ m agarose coating (8% agarose) on a 90-106 ⁇ m borosilicate core.
  • Example 3 Anion Exchange Bead (96-112 ⁇ m) with 90-106 ⁇ m borosilicate core and 3 ⁇ m coating.
  • Example 3 was made exactly as example 1, except the starting bead consisted of a 3 ⁇ m agarose coating (8% agarose) on a 90-106 ⁇ m borosilicate core.
  • Example 4 Anion Exchange Bead (114 ⁇ m) with 106 ⁇ m alkaline resistant (AR) glass core and 4 ⁇ m coating.
  • Agarose coated AR beads were made using a process described in Example 1.
  • the agarose coated beads (114 ⁇ m diameter, 4 ⁇ m coating, 8% agarose, 50 ml_ volume) were crosslinked using epichlorohydrin according to the method described in Example 1.
  • Anion exchange groups were added with the following method: 5OmL of beads were added to 50 mL of 75% glycidyl trimethylammonium chloride (GTMAC). To this mixture, 1.67 mL of 50% wt sodium hydroxide was added. The reaction was stirred at 35 0 C overnight. The beads were then filtered and washed with three 200 mL volumes of Milli-Q® water. The beads were stored in 20% ethanol/water solutions.
  • GTMAC glycidyl trimethylammonium chloride
  • Example 5 Anion Exchange Bead (80 ⁇ m) with 70 ⁇ m borosilicate core and 5 ⁇ m coating with polymeric ligand.
  • Agarose coated borosilicate beads were made using a process described in Example 1.
  • the agarose coated beads (34 mL) were crosslinked using epichlorohydrin according to the method described in Example 1.
  • a polyamine was added to the agrose coating using the following method: 34mL of beads were added to 11 mL of water, 12 mL of 50% wt sodium hydroxide and 2.5g Na 2 SO 4 . This mixture was stirred for 15 minutes and then 11 mL of allyl glycidyl ether was added. The reaction was stirred overnight at room temperature. The beads were then filtered and washed with three 200 mL volumes of Milli-Q® water.
  • the beads (34mL) were then equilibrated in a stirring solution of 4% sodium acetate buffer (60 mL). After equilibration, an aqueous solution of 4% sodium acetate with 1% wt bromine was added drop wise until a yellow color (residual bromine) persisted for more than 1 minute. The remaining bromine was neutralized with a 5% aqueous sodium formate solution.
  • the beads were then filtered and washed with three 200 mL volumes of Milli-Q® water. The beads (34 mL) were then added to 20 mL of water. To this mixture, 40 mL of 50% of polyethyleneimine (MW1200) was added.
  • Example 6 Anion Exchange Bead (80 ⁇ m) with 70 ⁇ m borosilicate core and 5 ⁇ m coating with polymeric ligand and multiple ligand addition steps.
  • Example 6 was made according to Example 5, except the anion exchange additional step (GTMAC) was repeated a second time.
  • GTMAC anion exchange additional step
  • Example 7 Determination of protein capacity. Dynamic capacity of BSA.
  • Anion exchange media were packed into omnifit columns and tested for dynamic binding capacity of a common benchmarking protein, bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the columns were equilibrated with 25 mmol Tris buffer (pH 8) and the protein (10g/L) was loaded onto the column with this same buffer.
  • the conductivity of the equilibration and loading buffer was varied using salt (NaCI) to achieve conductivities between 2mS (no additional salt) to 14 mS.
  • NaCI salt
  • the capacity at 10% breakthrough was used to compare the different media.
  • Figure 2 shows how the dynamic capacity for BSA varies with buffer ionic strength for Sepharose®-Q Fast Flow media and the media made according to Example 4.
  • FIG. 1 One of the target separations for flow-through anion exchange media is the removal of virus from the product protein.
  • a model virus feed system containing ⁇ X174 bacteriophage was used to investigate the viral removal properties of the media.
  • the media was challenged wit 10 7 pfu/mL of ⁇ X174 in 25 mmol Tris buffer (pH 8).
  • Figure 4 shows the viral removal (shown as Log removal) of the bead from Examples 1, 2 and 4 as compared to Q- Sepharose® FF media at 2 minute residence time (7 cm column run at 210 cm/hr). While example 4 has a slightly lower (but still >5 log) viral removal, Examples 1 and 2 gave essentially the same LRV as Q-Sepharose® FF media under the same conditions.
  • Example 4 It is also important to recognize that the improved permeability of the cored bead in Example 4 (>4x, see below) allows for the material to be packed into column bed heights greater than the 20 cm limit associate with Sepharose® media. If this advantage is exploited, a 40 cm column of Example 4 could be run at the same velocity and get an effective residence time of 4 minutes (where the log viral removal is 7 for Example 4, data not shown). Another possibility is to run the 40 cm bed height at twice the velocity, thus processing the material in half the time, but still maintaining the 2 minute residence time necessary for virus removal.
  • Example 9 Host cell protein (CHOP) removal performance of Pellicular or “Cored” bead media.
  • a Chinese hamster ovary cell line was used to generate a host cell protein feed solution.
  • the CHOP feed solution was fermented and clarified by a contract manufacturer.
  • a model CHOP feed stream was generated by purifying the clarified feed through a Prosep® A media (available from Millipore Corporation) column followed by buffer exchange and concentration by a tangential flow filtration step.
  • the model feed system contained 4 ng/mL of CHOP protein determined using a CHOP ELISA assay.
  • the model feed stream was processed in a "flow-through” mode with fractions collected for analysis every 10 column volumes.
  • the CHOP ELISA assay results for flow-through fractions are shown in Figure 5.
  • CHOP removal was the same for Example 4 and Q- Sepharose® FF media, even though Example 4 has 4 times the permeability of Q- Sepharose® FF media.
  • the CHOP removal for Example 1 is better than the removal observed for Q-Sepharose® FF media, at essentially the same permeability.
  • Example 10 Permeability of Pellicular or "Cored” bead media.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

L'invention concerne un procédé de purification de protéines à l'aide d'un lit de « billes à noyaux » dans un mode de chromatographie à écoulement ou de polissage. L'utilisation d'un milieu d'agarose pelliculaire/de « billes à noyaux » non poreuses assure un milieu qui possède une perméabilité et des propriétés mécaniques de beaucoup améliorées par rapport aux billes existantes, tout en maintenant un « facteur de sécurité » de protéine augmenté et une facilité de mise à l'échelle par rapport aux dispositifs de membrane existants. Les milieux d'agarose à noyaux utilisent des billes internes rigides (dans ce cas, des billes de polymère synthétique, de métal ou de verre non poreuses) qui forment des lits à garnissage incompressibles et hautement perméables, notamment lorsque le revêtement d'agarose est relativement mince (par exemple, 5-15 microns sur un noyau de diamètre supérieur à 50 microns). Cette perméabilité améliorée conduit à une capacité de protéines inférieure (notamment en termes de g de protéine/litre de milieu). Par conséquent, ces matières peuvent être utilisées dans des applications où des lits hautement perméables sont voulus en raison du fait que l'on peut régler la capacité, la perméabilité et la rigidité de lit pour augmenter au maximum le débit ou réduire au minimum le temps de traitement.
PCT/US2006/043135 2006-11-06 2006-11-06 Procédé pour une chromatographie à écoulement WO2008057074A1 (fr)

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Cited By (1)

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EP0266580A2 (fr) * 1986-11-03 1988-05-11 Excorim Kb Méthode pour enrober des particules solides d'un gel hydrophile et particules enrobées par cette méthode
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WO2000057982A1 (fr) * 1999-03-26 2000-10-05 Upfront Chromatography A/S Purification en lit fluidise de bio-macromolecules telles qu'un adn plasmidique, un adn chromosomique, un arn, un adn viral, des bacteries et des virus
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EP0266580A2 (fr) * 1986-11-03 1988-05-11 Excorim Kb Méthode pour enrober des particules solides d'un gel hydrophile et particules enrobées par cette méthode
EP0328256A1 (fr) * 1988-01-21 1989-08-16 Owens-Corning Fiberglas Corporation Fibres de verre recouvertes d'agarose servant de garnissage ou de milieu chromatographique pour colonne dans les bioséparations
WO2000057982A1 (fr) * 1999-03-26 2000-10-05 Upfront Chromatography A/S Purification en lit fluidise de bio-macromolecules telles qu'un adn plasmidique, un adn chromosomique, un arn, un adn viral, des bacteries et des virus
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MARTIN DEL VALLE E ET AL: "Use of ceramic monoliths as stationary phase in affinity chromatography", BIOTECHNOLOGY PROGRESS, vol. 19, no. 3, 2003, pages 921 - 927, XP002327160, ISSN: 8756-7938 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110081700A1 (en) * 2009-07-31 2011-04-07 Baxter Healthcare S.A. Methods of Purifying Recombinant Adamts13 and Other Proteins and Compositions Thereof
US8945895B2 (en) * 2009-07-31 2015-02-03 Baxter International Inc. Methods of purifying recombinant ADAMTS13 and other proteins and compositions thereof
US11661593B2 (en) 2009-07-31 2023-05-30 Takeda Pharmaceutical Company Limited Methods of purifying recombinant ADAMTS13 and other proteins and compositions thereof

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