WO2009091680A1 - Purification de protéines par chromatographie liquide haute performance (hplc) utilisant des résines semi-compressibles - Google Patents

Purification de protéines par chromatographie liquide haute performance (hplc) utilisant des résines semi-compressibles Download PDF

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Publication number
WO2009091680A1
WO2009091680A1 PCT/US2009/030692 US2009030692W WO2009091680A1 WO 2009091680 A1 WO2009091680 A1 WO 2009091680A1 US 2009030692 W US2009030692 W US 2009030692W WO 2009091680 A1 WO2009091680 A1 WO 2009091680A1
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column
resin
flow rate
protein
hplc
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PCT/US2009/030692
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English (en)
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David J. Roush
Joseph Nti-Gyabaah
Michael James Iammarino
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Merck & Co., Inc.
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Publication of WO2009091680A1 publication Critical patent/WO2009091680A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation

Definitions

  • the invention relates generally to a method for the purification of proteins from a fermentation broth using rigid and semi-compressible high performance liquid chromatographic (HPLC) resins to produce a purified drug substance.
  • HPLC high performance liquid chromatographic
  • the invention specifically provides a process for the purification of monoclonal antibodies.
  • Therapeutic proteins are typically produced by cell culture using either mammalian or bacterial cell lines engineered to express the protein of interest (POI) from a recombinant plasmid containing the gene encoding the protein. Separation of the expressed POI from the mixture of components needed to grow and maintain such cell lines and from any by-products of the cells themselves to a purity sufficient for use as a human therapeutic poses a daunting challenge to pharmaceutical manufacturers.
  • POI protein of interest
  • a typical purification process for an antibody includes an affinity-purification step, such as Protein A affinity chromatography, an anion exchange change (AEX) chromatography step and a cation exchange chromatography (CEX) step.
  • affinity-purification step such as Protein A affinity chromatography, an anion exchange change (AEX) chromatography step and a cation exchange chromatography (CEX) step.
  • Protein A affinity chromatography the most commonly used primary capture step for proteins and, in particular, monoclonal antibodies (mAb) is where a mAb in a mixture primarily and selectively binds to protein A via its Fc region and the impurities, such as host cell proteins (HCPs), DNA and endotoxins, remain unbound (Lancet et al, Biochem, and Biophvs. Res. Comm., 85(2): 608-614 (1978); Sulica et al. Immunology, 38(1): 173-179 (1979); Ghose et al, Biotechnol Prog.. 20(3): 830-840 (2004); Ghose et al., Biotechnol Bioeng.. 96(4): 768-779 (2007)).
  • HCPs host cell proteins
  • AEX and CEX are used as complimentary polishing steps in the industry to remove DNA, endotoxin and any leached protein A ligand, as well as HCPs.
  • CEX is also used to remove any aggregates in the product (Fahner et al., Biotechnol Genet. Eng, Rev.. 18: 301-327 (2001); Iyer et al. Bio. Pharm. Int. 15: 14-20 (2002); Shukla et al, J. Chrom. B, 848(1): 28-39 (2007)). While the available purification processes may achieve the desired purity standards, they often do so by a trade off in cost or productivity.
  • HPLC has been used for the purification of polypeptides and monoclonal antibodies.
  • Santucci, A., et al., J. Immunological Methods, 114: 181-185 (1988) purified an antibody using the antigen as an immobilized ligand bound to the HPLC column matrix. Purified antibody is recovered by lowering the pH of the elution buffer.
  • Pavlu, B., et al., J. Chromato graphy, 359: 449-460 (1986) purified an antibody in one chromatographic step by precipitating the antibody by ammonium sulphate prior to HPLC.
  • Burchiel, S., et al., 1 Immunological Methods, 69: 33-42 (1984) purified a murine antibody utilizing anion exchange and gel permeation chromatograph using HPLC under neutral pH conditions with a hydrophilic resin.
  • Production scale processes using protein A purification of monoclonal antibodies has typically focused on optimizing flow rates and column length in order to design bioprocesses for maximum production (Fahrner, R., et al., Bioprocess Engineering, 21 : 287-292 (1999).
  • Production scale processes have also used the performance characteristics of the sorbent to optimize productivity (Fahrner, R. et al., Biotechnol. Appl. Biochem., 30: 121-128 (1999), where differences in capacity and pressure drop affected the production rate.
  • Applicants herein have developed a platform process utilizing HPLC and semi- compressible resins for all modes of purification typically employed for the manufacture of a therapeutic protein.
  • the inventive method described herein is more cost effective and has improved productivity over prior methods employed therein.
  • the invention claimed herein is a process for purifying a protein of interest (POI) comprising:
  • the process optionally includes an equilibration step prior to the loading of the liquid mixture containing the POL Steps (a) through (d) may be repeated following regeneration of the column with a regeneration buffer so as to completely utilize the resin life.
  • the column is stainless steel.
  • the claimed process is a protein A affinity chromatography step, wherein said semi-compressible resin is selected from the group consisting of a polystyrene/divinyl benzene resin or a silica based resin.
  • the wash, elution, and regeneration of the column are carried out at a linear flow rate in excess of 720 cm/h, and the loading and elution of the liquid mixture containing the POI are carried out at a linear flow rate in excess of 720 cm/h.
  • the claimed process is an anion exchange chromatography step, wherein the semi-compressible resin is selected from the group consisting of a polyvinyl aery lam ide resin and a polystyrene/divinyl benzene resin coupled to a quaternary amine Hgand.
  • the claimed process is a cation exchange chromatography step, wherein the semi-compressible resin is selected from the group consisting of a polyvinyl acrylamide resin and a polystyrene/divinyl benzene resin coupled to a sulfopropyl based ligand.
  • the wash, elution, and regeneration of the column are carried out at a flow rate of about 3,000 cm/h, and the loading of the liquid mixture containing the POI are carried out at a flow rate in excess of 1 ,200 cm/h.
  • Figure 1 depicts the pressure/flow curve for a silica-Protein A resin (Asahi Glass SI-Tech, Kobe, Japan).
  • Figure 2 depicts the pressure/flow curve for a POROS 5OA Protein A resin
  • Figure 3 depicts the pressure/flow curve for a UNOsphere Q anion exchange resin (BioRad, Hercules, Pa),
  • Figure 4 depicts the pressure/flow curve for a POROS HQ50 anion exchange resin (Applied Biosystems, Foster City, CA).
  • Figure 5 depicts the pressure/flow curve for a UNOsphere S cation exchange resin BioRad, Hercules, CA).
  • Figure 6 depicts the pressure/flow curve for a POROS HS50 cation exchange resin (Applied Biosystems, Foster City, CA).
  • Figure 7 depicts the yield (%) and productivity (kg POI/L-resin/hour) as a function of loading linear velocity for an Asahi silica-protein A resin (Asahi Glass SI-Tech, Kobe, Japan).
  • Figure 8 depicts the yield (%) and productivity (kg POI/L-resin/hour) as a function of loading linear velocity for a POROS 5OA protein A resin (Applied Biosystems, Foster City, CA).
  • Figure 9 depicts the yield (%) and productivity (kg POI/L-resin/hour) as a function of loading linear velocity for an UnoSphere Q AEX resin BioRad, Hercules, CA).
  • Figure 10 depicts the yield (%) and productivity (kg POI/L-resin/hour) as a function of loading linear velocity for a POROS 50HQ AEX resin (Applied Biosystems, Foster City, CA).
  • Figure 1 1 depicts the yield (%) and productivity (kg POI/L-resin/hour) as a function of loading linear velocity for an UnoSphere S CEX resin (BioRad, Hercules, CA).
  • Figure 12 depicts the yield (%) and productivity (kg POI/L-resin/hour) as a function of loading linear velocity for a POROS HS50 CEX resin (Applied Biosystems, Foster City, CA).
  • protein is used herein in the broadest sense and includes polypeptides made up of amino acid residues covalently linked together by peptide bonds.
  • antibody is used herein in the broadest sense and specifically refers to monoclonal antibodies or fragments thereof.
  • medium pressure or “moderate pressure” when referring to high performance liquid chromatography herein means a pressure of about 15 psi to 120 psi.
  • high pressure when referring to high performance liquid chromatography herein means a pressure of about 120 psi to 5000 psi.
  • high performance liquid chromatography column herein refers to a column that can withstand of up to 1000 psi.
  • si-compressible resin refers to a mechanically stable resin, that is, one that does not irreversibly compress, collapse, or irreversibly yield to pressure stress of 200 psi (i.e. 200 pounds-force per square inch). This means that the resin does not permanently deform as a result of the application of 200 psi pressure.
  • An example of a pressure flow curve for Capto Q 5 a commercial resin that exhibits this properly up to 50 psi, is presented in Wang, J. Chromatography A, 1155 (1): 74-84 (2007).
  • high salt buffer refers to a buffer that contains salt in the range of about 0.2 to 1.0 M NaCl, see for example, Tugcu et al, Biotechnology and Bioengineering. 99(3): 599-613 (2008).
  • concentration buffer or “column conditioning buffer” as used herein refers to the buffer used to condition the column before the product is loaded onto the column, for example, a 25 mM sodium phosphate buffer used to condition the protein column before the feed is loaded can be an equilibration buffer, Tugcu et al. s Biotechnology_and Bioengineering, 99(3): 599-613 (2008).
  • regeneration buffer refers to the buffer used to clean the column to remove bound impurities, for example, a high salt buffer, a NaOH-containing, a detergent-containing, or a phosphoric acid-containing buffer, Tugcu et al., Biotechnology and Bioengineering. 99: 599-613 (2007).
  • column volume or “CV” as used herein refers to the volume of packed resin inside the column including any void volume. For example, if a 10 L column is packed with 2 L of resin, one CV is 2 L.
  • load flow rate refers to the volumetric flow rate at which the protein of interest (POI) is loaded onto the column.
  • flow rate or “linear flow rate” or “linear flow velocity (LV)” as used herein refers to the volumetric flow rate divided by the packed bed (or internal column) cross sectional area.
  • productivity refers to the amount of purified product, i.e. POI, recovered per quantity of chromatographic resin in a given time period, for example, kg POI purified/liter resin/hour.
  • yield is the of amount product recovered divided by the amount of product loaded into the column multiplied by 100. For example, a column loaded with a solution that contained 10Og of product, but from which 9Og of product was recovered from the elution stream, would have a 90% yield.
  • HPLC platform process refers to one or more steps, phases or modes, used to purify a protein of interest (POI), including protein A affinity chromatography, anion exchange chromatography and cation exchange chromatography, carried out via HPLC.
  • POI protein of interest
  • AEX anion exchange
  • chromatography refers to a process such as that of Fahner et al. s Biotechnology and Genetic Engineering Reviews, 18: 301-327 (2001) or Tugcu et al., Biotechnology and Bioengineering, 99(3): 599-613 (2008), in which a quaternary amine ligand, coupled to a base matrix composed of agarose such as Q Sepharose Fast Flow (GE Healthcare, Piscataway, NJ) or acrylamido and vinylic monomers, such as UNOsphere Q (BioRad, Hercules, CA), is packed into a BPG glass column (GE Healthcare, Piscataway, NJ) and washed with a high salt buffer at 300 cm/h.
  • agarose such as Q Sepharose Fast Flow
  • acrylamido and vinylic monomers such as UNOsphere Q (BioRad, Hercules, CA)
  • UNOsphere S BioRad, Hercules, CA
  • UNOsphere S BioRad, Hercules, CA
  • the bed is then equilibrated with an equilibration buffer and the product is loaded.
  • the column is again washed with the equilibration buffer, eluted, and regenerated using NaOH. Because of column pressure limitations (maximum of 45 psi), the linear flow rate to the column for all steps herein cannot exceed 300 cm/h.
  • a protein purification process typically comprises three chromatographic stages that ultimately yield the final drug substance, including, affinity (protein A), cation exchange (CEX), and anion exchange (AEX). While Applicants have present them herein in a specific sequence, those of ordinary skill in the art would understand and know how to modify the sequence of the stages for each particular application.
  • HPLC can provide advantages with respect to speed and resolution relative to other chromatographic systems. It allows the user to more accurately control the chromatographic conditions, which may result in a higher degree of reproducibility over a wide range of volumes and concentrations as compared to other forms of chromatography.
  • HPLC can provide a relatively fast purification with a reduced dilution of recovered product compared to conventional methods.
  • the conventional HPLC process has not been used on a large scale for each stage of a protein purification process because of pressure limitations imposed by available columns, which typically only allow use up to 7 atm or about 120 psi, and manufacturer recommended limits for available resins, that limit the pressure to below 3 atm or about 45 psi.
  • Other disadvantages include the lack of commercially available preparative off-the-shell HPLC equipment, and high pressure column hardware.
  • Piscataway, NJ has a recommended pressures of 45 psi or less.
  • the conventional belief by those of ordinary skill in the art was that use outside the recommended operating pressures would result in the collapse or failure of the resin bed, with a resulting loss of velocity and productivity.
  • optimization of the conventional processes focused on feed loading flow rates, which were typically kept to a maximum of about 300 cm/h for a 20 cm packed resin bed height.
  • Applicants herein have developed a large scale platform process for purifying a protein and, in particular, an antibody, using HPLC for each stage of the purification process.
  • the claimed process may use one or more steps to carry out the purification.
  • the inventive methods combine the use of a stainless steel HPLC column with a semi- compressible resin, such as a polyacrylamide or a polystyrene based resin, to produce a process that operates at higher pressures, with increased flow rates and reduced cycle times than conventional protein purification processes. While the specific methods herein utilize different semi-compressible resins, the use of a common type of resin allows for process efficiencies in the selection of columns and the use of a single pump skid/process platform, or single unit operation.
  • Example IA The protocol for the evaluation of the protein A HPLC resins is set forth in Example IA.
  • loading was evaluated at a fixed bed height (25 cm) and a fixed loading rate (3Og of product per liter of resin).
  • product recovery was comparable to that from the conventional process.
  • the primary purification step for the production of high purity therapeutic proteins and, specifically monoclonal antibodies (mAb), is protein A affinity chromatography.
  • Various approaches for increasing productivity for protein A affinity chromatography using medium pressure chromatography have been studied. See for example, the use of a polystyrene divinyl benzene resin (POROS protein A) in a protein A purification, Fahner et al., Bioprocess Engineering, 21: 287-292 (1999), where due to pressure limitations (of about 30 psi) of their system (acrylic and glass columns) it was necessary to reduce the length of their packed bed (bed height) in order to increase the linear flow rate to an acceptable level, Fahrner et al., Biotechnol. Appl. Biochem., 30: 121-128 (1999).
  • POROS protein A polystyrene divinyl benzene resin
  • a glass column, packed with MabSelectTM the industry standard for this type of purification, is equilibrated with 5 column volumes (CV) of an equilibration buffer at a linear flow rate of 150 to 300 cm/h using a 25 cm packed-resin column. Thereafter, the cell culture supernatant (product stream) is loaded onto the column using a linear flow rate of 150 to 300 cm/h.
  • the column Upon completion of loading the feed, the column is washed with 5 column volumes (CV) of the equilibration buffer at 150 to 300 cm/h rate and the product is eluted at 150 to 300 cm/h, after which the column is regenerated with 5 column volumes (CV) of regeneration buffer at 150 to 300 cm/h.
  • CV column volumes
  • Applicants herein have developed a method for protein A affinity chromatography that solves the aforementioned issues through the use of a semi-compressible resin in a high- performance-liquid-chromatography (HPLC) column.
  • HPLC high- performance-liquid-chromatography
  • Applicants have found unexpectedly that semi-compressible resins, such as silica protein A (Asahi Glass SI-Tech, Kobe, Japan) or POROS ® 50A (Applied Biosystems, Foster City, CA) can be utilized outside conventional operating pressures and loading rates in an HPLC column to carry out protein A affinity chromatography without the loss of resin integrity, i.e. collapse, and resulting loss of flow rate. See Example 2.
  • resins of this type are used in the claimed protein A HPLC method at flow rates of about 3,000 cm/h to 8,000 cm/h, corresponding to pressures from about 200 psi to 1 ,300 psi, more preferably at flow rates of about 4,000 cm/h to 7,000 cm/h, corresponding to pressures of about 600 psi to 1200 psi, and most preferably at flow rates of about 5,000 cm/h to 6,000 cm/h 5 corresponding to pressures of about 800 psi to 1000 psi.
  • inventive method allows one of ordinary skill in the art to more accurately control the chromatographic conditions, resulting in a higher degree of reproducibility over a wide range of volumes and concentrations, as compared to other forms of chromatography.
  • inventive method also provides a relatively fast purification with reduced dilution of recovered product as compared to other forms of chromatography, such as low or medium pressure chromatography.
  • Table 4A and 4B show the performance of the inventive HPLC method, using the Asahi silica-protein A and the POROS ® 50A resins, respectively, relative to a conventional protein A process. Yield and product quality were comparable to that achieved with the conventional process. Applicants have found unexpectedly that an HPLC process could be carried out for this stage of the protein purification and with linear flow rates significantly outside the conventional range, which resulted in an over ten-fold increase in overall productivity as compared to the conventional protein A process. As such, the increased linear flow rate would allow one to complete chromatographic purification of a batch about ten times faster than the conventional process.
  • the equilibration step was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the
  • the loading step was carried out at a linear flow rate of 720 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the loading
  • the chase wash was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the chase
  • the elution step was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the elution
  • the regeneration #1 step was carried out at a linear flow rate of 5 ⁇ 000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that
  • the equilibration step was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the
  • the loading step was carried out at a linear flow rate of 720 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the loading
  • the chase wash was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the chase
  • the elution step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that the elution
  • the regeneration #1 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 300 cm/h (for the conventional process). This means the that
  • the cumulative impact of increasing the linear flow rates for all steps (including the loading step) for the inventive process translates to completing the purification process for the protein A step in about four to five times faster than the conventional process.
  • the bed Upon completion of the loading, the bed is washed with 5 CV of equilibration buffer at the same flow rate, following which the bed is regenerated with 5 CV of NaOH at the same flow rate. Because of column pressure limitations (maximum of 45 psi), the linear flow rate has to be maintained at 300 cm/h or less for each of the elution steps. The system operates in a flow-through mode so that the product is not retained, which reduces the level of any exogenous DNA and HCP further.
  • Applicants have developed a method for AEX that utilizes semi-compressible resins in high performance liquid chromatography (HPLC) columns operated in excess of 200 psi.
  • HPLC high performance liquid chromatography
  • Applicants have found unexpectedly that semi- compressible resins, such as UNOsphere Q (BioRad, Hercules, CA) and POROS ® HQ50 (Applied Biosytems, Foster City, CA) can be utilized outside conventional operating pressures and loading rates in an HPLC column to carry out an AEX chromatography without the loss of resin integrity. See Example 3.
  • the resins were used in the claimed AEX HPLC method at flow rates of about 1 ,000 cm/h to 8,000 cm/h, corresponding to pressures from about 100 psi to 1,300 psi, more preferably at flow rate of about 4,000 cm/h to 8,000 cm/h, corresponding to pressures from about 150 psi to 1,300 psi, and most preferably at flow rates of about 5,000 cm/h to 7,000 cm/h, corresponding to pressures from about 180 psi to 1,150 psi.
  • the flow rate of 5,000 cm/h was used for the equilibration, wash and regeneration steps, as compared to 450 cm/h in the conventional process, resulting in significantly faster flow rates (5,000 cm/h) overall, i.e. at each step of the process, and higher operating pressures (200 psi) than the conventional protocol.
  • the use of a semi-compressible resin overcomes the pressure limitation of the conventional process such that the resin bed height could be the same as that of the conventional process or even be increased, while maintaining (or increasing) the flow rate. These operating conditions translate into increased speed, i.e. a faster linear flow rate without pressure concerns, with similar resolution of impurities as a result of reduced axial and radial dispersions.
  • Tables 5 A and 5B shows a comparison of the operating conditions for the inventive AEX HPLC method using UNOsphere Q and POROS ® HQ50, respectively, relative to a convention AEX process.
  • the use of the inventive method allows one of ordinary skill in the art to more accurately control the chromatographic conditions, resulting in a higher degree of reproducibility over a wide range of volumes and concentrations as compared to other forms of chromatography.
  • the inventive method also provides a relatively fast purification with a reduced dilution of recovered product as compared to other forms of chromatography, such as low or medium pressure chromatography.
  • the equilibration step was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the
  • the loading step was carried out at a linear flow rate of 1 ,800 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the loading
  • the chase wash was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the chase wash
  • the elution step was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the elution step
  • the regeneration # 1 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the
  • the regeneration #2 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the
  • the cumulative impact of increasing the linear flow rates for all steps (including the loading step) for the inventive process translates to completion of the purification process for the AEX step in about four to five times faster than the conventional process.
  • the equilibration step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the
  • the chase wash was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared Io 450 cm/h (for the conventional process). This means that the chase wash
  • the elution step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the elution step
  • the regeneration # 1 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the
  • the regeneration #2 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the
  • the cumulative impact of increasing the linear flow rates for all steps (including the loading step) for the inventive process translates to finishing up the purification process for the AEX step in about four to five times faster than the conventional process.
  • a sulfopropyl-based ligand coupled to a base matrix composed of polystyrene/divinylbenzene, such as POROS ® HS50 (Applied Biosystems, Foster City, CA), or acrylamido and vinylic monomers, such as UNOsphere S (BioRad, Hercules, CA) is packed into a glass column and washed with 5 CV of high salt buffer at a standard linear flow rate of 450 cm/h. Thereafter, the column is equilibrated with 8 CV of equilibration buffer. The product is then loaded at the standard flow rate.
  • POROS ® HS50 Applied Biosystems, Foster City, CA
  • UNOsphere S BioRad, Hercules, CA
  • the resins were used in the claimed CEX HPLC method at flow rates of about 1,000 cm/h to 8,000 cm/h, corresponding to pressures from about 100 psi to 1,200 psi, more preferably at flow rate of about 4,000 cm/h to 8,000 cm/h, corresponding to pressures from about 150 psi to 1,200 psi, and most preferably at flow rates of about 5,000 cm/h to 6,000 cm/h, corresponding to pressures from about 180 psi to 1,000 psi.
  • the flow rate of 5,000 cm/h was used for the equilibration, wash and regeneration steps, as compared to 450 cm/h in the conventional process, resulting in significantly faster flow rates (5,000 cm/h) overall, i.e. at each step of the process, and higher operating pressures (200 psi) than the conventional protocol.
  • Tables 6 A and 6B show a comparison of the operating conditions for the inventive CEX HPLC process using UNOshpere S (BioRad, Hercules, CA) and POROS ® HS50 (Applied Biosystems, Foster City, CA), respectively, relative to a conventional CEX process.
  • the increased flow for the inventive process allows one to complete the chromatographic purification of a batch more than four times faster than the conventional process.
  • the equilibration step was carried out at a linear flow rate of 5,000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the
  • the loading step was carried out at a linear flow rate of 1,800 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the loading step
  • the elution step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the elution step
  • the regeneration #1 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the
  • the regeneration #2 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the
  • the cumulative impact of increasing the linear flow rates for all steps (including the loading step) for the inventive process translates to completion of the purification process for the AEX step four to five times faster than the conventional process.
  • the equilibration step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the
  • the chase wash was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the chase wash
  • the elution step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process TM compared to 450 cm/h (for the conventional process). This means that the elution step
  • the regeneration #1 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the
  • the regeneration #2 step was carried out at a linear flow rate of 5000 cm/h (for the HPLC process - compared to 450 cm/h (for the conventional process). This means that the
  • the cumulative impact of increasing the linear flow rates for all steps (including the loading step) for the inventive process translates to completion of the purification process for the CEX step eight to nine times faster than the conventional process.
  • an HPLC process can be carried out for each stage of protein purification and that such processes can be carried using a semi-compressible resin in a stainless steel column significantly outside conventional process limits.
  • the inventive platform process unexpectedly resulted in significantly increased flow rates with corresponding significant reductions in overall cycle time and increases in productivity. In each stage, product quality either met or exceeded industry standards.
  • the claimed process offers advantages relative to the conventional process by providing cost effective use of the resins, reduced cycle times,, increased productivity and an overall lower unit cost for protein purification.
  • the inventive process allowed Applicants to complete the protein purification in about five hours, as compared to about 40 hours for the conventional process. This translates to about an eight-fold increase in productivity for the HPLC process.
  • the evaluation involved using a Waters FractionLynxTM HPLC System HPLC (Waters Corporation, Milford, MA) system to pump phosphate buffer (PBS) 10 mM sodium phosphate, 100 mM NaCl, pH 7.2 (Hyclone, Logan, UT) through each column at different flow rate and monitoring the pressures.
  • PBS phosphate buffer
  • PBS Phosphate buffer
  • 10 mM sodium phosphate, 100 mM NaCl, pH 7.2 were purchased from Hyclone, Logan, UT.
  • Buffer components: citric acid and citrate trihydrate were purchased from Fisher Scientific (Pittsburg, PA) and sodium phosphate dibasic and monobasic, Tris-HCl, Trizmabase, and 5 M NaCl solution were purchased from Sigma (St. Louis, MO).
  • PBS Phosphate buffer
  • 25 mM sodium phosphate, pH 7.5 were purchased from Hyclone, Logan, UT. 5 M NaCl solution were purchased from Sigma (St. Louis, MO).
  • Analytical protein A HPLC analysis of samples was performed on an Agilent 1100 HPLC system (Agilent, Palo Alto, CA) equipped with a POROS PA ID immunoaffinity cartridge. Chromatography experiments were performed using Waters FractionLynx HPLC System. DNA and host cell protein (HCP) levels in the product pools were tested using the Picogreen assay Guilloa et al., J, of Chromatography A, (11 13) Issues 1-2: 239-243 (2006).
  • HCP host cell protein
  • an anti-ADDL mAb (WO 2006/055178) was employed to demonstrate the inventive process.
  • PBS Phosphate buffer
  • 50 mM citrate buffer, pH 4.5 were purchased from Hyclone, Logan, UT. 5 M NaCl solution were purchased from Sigma (St. Louis, MO).
  • Analytical protein A HPLC analysis of samples was performed on an Agilent 1 100 HPLC system (Agilent, Palo Alto, CA) equipped with a POROS PA ID immunoaffinity cartridge. Chromatography experiments were performed using Waters FractionLynx HPLCTM System (Waters Corporation, Milford, MA). DNA and host cell protein (HCP) levels in the product pools were tested using the Picogreen assay Guilloa et al., J. of Chromatography A (1 113) Issues 1-2: 239-243 (2006).
  • HCP host cell protein

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention porte sur un procédé de purification d'une protéine et, en particulier, d'un acide monoclonal, lequel procédé met en jeu l'utilisation d'une résine semi-compressible dans une colonne de chromatographie liquide haute performance actionnée à des débits élevés et des pressions de fonctionnement élevées. Le procédé peut être utilisé pour tous les stades de purification des protéines incluant la chromatographie d'affinité de protéine A, une chromatographie d'échange d'anions et une chromatographie d'échange de cations.
PCT/US2009/030692 2008-01-15 2009-01-12 Purification de protéines par chromatographie liquide haute performance (hplc) utilisant des résines semi-compressibles WO2009091680A1 (fr)

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Cited By (2)

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US9488625B2 (en) 2010-12-15 2016-11-08 Baxalta GmbH Purification of factor VIII using a conductivity gradient
US11155575B2 (en) 2018-03-21 2021-10-26 Waters Technologies Corporation Non-antibody high-affinity-based sample preparation, sorbent, devices and methods

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9488625B2 (en) 2010-12-15 2016-11-08 Baxalta GmbH Purification of factor VIII using a conductivity gradient
US11155575B2 (en) 2018-03-21 2021-10-26 Waters Technologies Corporation Non-antibody high-affinity-based sample preparation, sorbent, devices and methods

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