US20060030696A1 - Protein a chromatography - Google Patents

Protein a chromatography Download PDF

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US20060030696A1
US20060030696A1 US11/210,669 US21066905A US2006030696A1 US 20060030696 A1 US20060030696 A1 US 20060030696A1 US 21066905 A US21066905 A US 21066905A US 2006030696 A1 US2006030696 A1 US 2006030696A1
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antibody
protein
igg
binding
purified
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Julian Bonnerjea
Anna Preneta
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Lonza Biologics PLC
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Priority claimed from PCT/EP2004/002041 external-priority patent/WO2004076485A1/en
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Priority to US11/419,306 priority Critical patent/US7847071B2/en
Priority to US12/910,301 priority patent/US20110040075A1/en
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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
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • 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
    • 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
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier

Definitions

  • the present invention relates to the field of antibody purification in biotechnological production. It is an object of the present invention to describe a novel process for purification of such antibody.
  • Protein A chromatography is widely used in industrial manufacturing of antibodies since allowing for almost complete purification of antibodies, that is usually IgG, in a single step from cell culture supernatants. Protein A affinity columns inevitably are subject to some degree of leakage of ligand from the column upon repeated runs. Partly, this may be due to proteolytic clipping of protein A from the column; in industrial manufacture of antibody for pharmaceutical applications, no protease inhibitor cocktails may be added for regulatory reasons. Unfortunately, this protein A or protein A fragment contaminants retain their affinity for IgG and are difficult to remove from the purified antibody due to ongoing complex formation.
  • Balint et al. demonstrated that such IgG-Protein complexes can be separated from uncomplexed IgG by gel filtration. Low through-put and loss in antibody yield are the disadvantages of this method.
  • U.S. Pat. No. 4,983,722 teaches selective separation of contaminating protein A from a protein-A purified antibody preparation by absorbing the mixture to an anion exchanger material and to separate both components by sequentially eluting the antibodies and protein A under conditions of increasing ionic strength. This resolution method is highly dependent on the pI of the antibody which is specific and highly variable for a given antibody. Further, throughput is limited by the steepness of the salt gradient required for obtaining separation.
  • FIG. 1 shows the result of pretreatment by means of non-reducing 10% SDS-PAGE for a staphylococcal protein A standard (lane 1: native protein A; lane 2: after pretreatment) and pure, uncoupled StreamlineTM recombinant protein A (provided by courtesy of Pharmacia, now Amersham-Biosciences; lane 4: native recombinant protein A; lane 5: after pretreatment).
  • FIG. 2 shows the levels of leaked recombinant protein A in antibody eluates from StreamlineTM recombinant protein A chromatography with single-point attached protein A in thioether linkage.
  • FIG. 3 further provides data on insubstantially reduced leakage of contaminant protein A during repeated runs of the protein A affinity chromatography.
  • FIG. 4 shows results of fractionation of aggregates during step elution of the antibody peak, as described further herein.
  • FIG. 5 shows outline of separation schemes, as further described herein.
  • a method of purifying an antibody comprises the steps of: firstly, purifying an antibody by means of protein A affinity chromatography wherein the protein A is a native protein A or a functional derivative thereof.
  • the purified antibody on an ion exchange material under conditions which allow for binding of the protein A or its functional derivative and thirdly, collecting the antibody, preferably collecting at least 70%, more preferably collecting at least 80%, most preferably collecting at least 90% of the amount of antibody loaded onto the ion exchange material in the flow-through of the ion exchanger whilst any contaminant protein A or protein A derivative is bound to the ion exchange material.
  • Protein A is a cell surface protein found in Staphylococcus aureus . It has the property of binding the Fc region of a mammalian antibody, in particular of IgG class antibodies. Within a given class of antibodies, the affinity slightly varies with regard to species origin and antibody subclass or allotype (reviewed in Surolia, A. et al., 1982, Protein A: Nature's universal ‘antibody’, TIBS 7, 74-76; Langone et al., 1982, Protein A of staphylococcus aureus and related immunoglobulin receptors, Advances in Immunology 32:157-252). Protein A can be isolated directly from cultures of S. aureus that are secreting protein A or is more conveniently recombinantly expressed in E.
  • coli (Lofdahl et al., 1983, Proc. Natl. Acad. Sci. USA 80:697-701). Its use for purification of antibodies, in particular monoclonal IgG, is amply described in the prior art (e.g. Langone et al., supra; Hjelm et al, 1972; FEBS Lett. 28: 73-76).
  • protein A affinity chromatography protein A is coupled to a solid matrix such as crosslinked, uncharged agarose (Sepharose, freed from charged fraction of natural agarose), trisacryl, crosslinked dextrane or silica-based materials. Methods for such are commonly known in the art, e.g.
  • Protein A binds with high affinity and high specificity to the Fc portion of IgG, that is the C ⁇ 2-C ⁇ 3 interface region of IgG as described in Langone et al., 1982, supra. In particular, it binds strongly to the human allotypes or subclasses IgG1, IgG2, IgG3 and the mouse allotypes or subclasses IgG2a, IgG2b, IgG3. Protein A also exhibits an affinity for the Fab region of immunoglobulins that are encoded by the V H gene family, V H III (Sasso et al., 1991, J.
  • IgG-binding domains of protein A bind to IgG via the Fc region, involving e.g. in human IgG-Fc residues 252-254, 433-435 and 311, as shown for the crystal structure in Deisenhofer et al. (1981, Biochemistry 20: 2361-2370) and in Sauer-Eriksson et al. (1995, Structure 3: 265-278) in case of the B-domain of protein A.
  • the finding of two essentially contiguous main binding sites in the Fc portion has been confirmed in the NMR-solution study of Gouda et al., 1998, Biochemsitry 37: 129-136.
  • each of the IgG-binding domains A to E of protein A is sufficient for binding to the Fc-portion of an IgG.
  • An IgG antibody according to the present invention is to be understood as an antibody of such allotype that it can be bound to protein A in a high-affinity mode. Further, apart from the Fc portions of the antibody that are relevant for binding to protein A, such antibody must not correspond to a naturally occuring antibody. In particular in its variable chain regions portions, it can be an engineered chimeric or CDR-grafted antibody as are routinely devised in the art.
  • An IgG-antibody according to the present invention is to be understood as an IgG-type antibody, in short.
  • An interaction compliant with such value for the binding constant is termed ‘high affinity binding’ in the present context.
  • such functional derivative of protein A comprises at least part of a functional IgG binding domain of wild-type protein A which domain is selected from the natural domains E, D, A, B, C or engineered muteins thereof which have retained IgG binding functionality.
  • An example of such is the functional 59 aminoacid ‘Z’-fragment of domain B of protein A which domain may be used for antibody purification as set forth in U.S. Pat. No.
  • an antibody binding fragment according to the present invention comprises at least two intact Fc binding domains as defined in this paragraph.
  • An example of such are the recombinant protein A sequences disclosed e.g. in EP-282 308 and EP-284 368, both from Repligen Corporation.
  • Protein A fragments that are engineered to allow of single-point attachement.
  • Single point attachment means that the protein moiety is attached via a single covalent bond to a chromatographic support material of the protein A affinity chromatography.
  • Such single-point attachment by means of suitably reactive residues which further are ideally placed at an exposed amino acid position, namely in a loop, close to the N- or C-terminus or elsewhere on the outer circumference of the protein fold.
  • Suitable reactive groups are e.g. sulfhydryl or amino functions. More preferably, such recombinant protein A or functional fragment thereof comprises a cysteine in its amino acid sequence.
  • the cysteine is comprised in a segment that consists of the last 30 amino acids of the C-terminus of the amino acid sequence of the recombinant protein A or functional fragment thereof.
  • the recombinant protein A or functional fragment thereof is attached by at least 50% via a thioether sulphur bond to the chromatographic support or matrix material of the protein A-affinity chromatography medium.
  • An example of such an embodiment is described e.g. in U.S. Pat. No. 6,399,750 from Pharmacia and is commercially available under the brand names of StreamlineTM or MabSelectTM from Amersham-Biosciences, depending on the nature of the support matrix used.
  • thioether is to be understood narrowly as a —S— bonding scheme irrespective of chemical context, deviating in this regard from normal chemical language; it is possible, for instance, that said —S— ‘thioether’ bridge according to the present invention is part of a larger functional group such as e.g. a thioester or a mixed acetal, deviating in this regard in the context of the present application from the reacitivity-based normal language of chemists.
  • the thioether bridge is a thioether bridge in its ordinary, narrow chemical meaning.
  • Such bridging thioether group can be e.g.
  • the protein A or functional protein A derivative according to the present invention is the recombinant protein A disclosed in U.S. Pat. No. 6,399,750 which comprises a juxtaterminal, engineered cysteine residue and is, preferably by at least 50%, more preferably by at least 70%, coupled to the chromatographic support material through the sulphur atom of said cysteine residue as the sole point of attachment.
  • such coupling has been achieved by means of epoxide mediated activation, more preferably either by means of 1,4-bis-(2,3-epoxypropoxy) butane activation of e.g.
  • an agarose matrix such as Sepharose Fast Flow (agarose beads crosslinked with epichlorohydrin, Amersham Biosciences, UK) or by means of epichlorohydrin activation of e.g. an agarose matrix such as Sepharose FF.
  • the first ion exchanger is an anion exchanger, in particular a quaternary amine-based anion exchanger such as Sepharose QTM FF (Amersham-Biosciences/Pharmacia), most preferably it is an anion exchanger having the functional exchanger group Q coupled to a matrix support which group Q is N,N,N-Trimethylamino-methyl, most preferably the anion exchanger is Sepharose QTM FF from Pharmacia/Amersham Biosciences.
  • the quarternary amino group is a strong exchanger which further is not susceptible to changes in pH of the loading/wash buffer.
  • the fast flow exchanger matrix is based on 45-165 ⁇ m agarose beads having a high degree of crosslinking for higher physical stability; further sepharose is devoid of the charged, sulfated molecule fraction of natural agarose and does not allow for unspecific matrix adsorption of antibody, even under condition of high antibody loads.
  • An example of such an embodiment can be found in the experimental section.
  • a contaminant protein A according to the present invention is any type of functional, IgG binding offspring of a protein A or a functional derivative thereof as defined above which is obtained upon eluting bound antibody from a protein A affinity chromatography column.
  • Such contaminant protein A species may result e.g. from hydrolysis of peptide bonds which is very likely to occur by means of enzyme action in particular in industrial manufacturing.
  • Protein A chromatography is applied as an early step in downstream processing when the crudely purified, fresh product solution still harbors considerable protease activity.
  • Dying cells in the cell culture broth or cells disrupted in initial centrifugation or filtration steps are likely to have set free proteases; for regulatory purposes, supplementation of the cell culture broth with protease inhibitors prior or in the course of downstream processing is usually not accomplished, in contrast to biochemical research practice.
  • Examples are Phenyl-methyl-sulfonyl-chloride (PMSF) or ⁇ -caproic acid.
  • PMSF Phenyl-methyl-sulfonyl-chloride
  • ⁇ -caproic acid Such chemical agents are undesirable as an additives in the production of biopharmaceuticals.
  • recombinant functional derivatives or fragments of protein A are less protease resistant than wild-type protein A, depending on the tertiary structure of the protein fold.
  • Amino acid segments linking individual IgG binding domains might be exposed once the total number of binding domains is reduced. Interdomain contacts may possible contribute to the stability of domain folding. It might also be that binding of antibody by protein A or said functional derivatives thereof influences or facilitates susceptibility to protease action, due to conformational changes induced upon binding of the antibody. Again, wild-type or full length protein A or functional, engineered fragments thereof might behave differently.
  • contaminant protein A according to the present invention still is functional, IgG binding protein and thus is associated with the protein A-purified antibody when loaded onto the subsequent ion exchange separation medium according to the present invention. The high-affinity based association of contaminant protein A with the purified antibody is the reason why it is difficult to efficiently separate contaminant protein A from purified antibody.
  • the antibody sought to be purified is harvested from a cell culture prior to purifying the antibody be means of protein A affinity chromatography.
  • said cell culture is a mammalian cell culture. Mammalian cells have large compartments called lysosomes harboring degradating enyzmes which are disrupted upon cell death or harvest.
  • said cell culture may be a myeloma cell culture such as e.g. NSO cells (Galfre, G. and Milstein, C. Methods Enzymology, 1981, 73,3).
  • Myeloma cells are plasmacytoma cells, i.e. cells of lymphoid cell lineage.
  • An exemplary NSO cell line is e.g.
  • NSO cell line ECACC No. 85110503, freely available from the European Collection of Cell Cultures (ECACC), Centre for Applied Microbiology & Research, Salisbury, Wiltshire SP4 0JG, United Kingdom.
  • ECACC European Collection of Cell Cultures
  • NSO cells have been found able to give rise to extremly high product yields, in particular if used for production of recombinant antibodies.
  • NSO cells have been found to give reproducibly rise to much higher levels of contaminant protein A than other host cell types at least with certain protein A affinity chromatography systems employing recombinant, shortened fragments of wild-type protein A which recombinant protein A is possibly single-point attached protein A.
  • StreamlineTM rProtein A affinity chromatography resin (Amersham Biosciences; essentially thioester single-point attached recombinant protein A as described in U.S. Pat. No. 6,399,750).
  • Levels of about or in excess of 1000 ng contaminant protein A/mg antibody could be obtained with StreamlineTM rProtein A affinity columns.
  • the method of the present invention distinguishes from the prior art in efficiently reducing contaminant protein A from such elevated levels to ⁇ 1 ng/mg antibody in a single, fast purification step, that is with a purification factor of about 1000 ⁇ .
  • the antibody that is to be purified by means of protein A affinity chromatography is not treated as to inactivate proteases at or after harvest, more preferably is not in admixture with at least one exogenously supplemented protease inhibitor after harvest.
  • said protease inhibitor is selected from the group consisting of PMSF, specific proteinase inhibiting peptides as described in Laskowski et al., 1980, Protein inhibitors of proteinases, Ann. Rev. Biochem. 49, 593-626, and epsilon-caproic acid.
  • the contaminant protein A is reduced to a concentration of ⁇ 10 ng/mg antibody, more preferably ⁇ 4 ng/mg antibody, most preferably ⁇ 1 ng/mg antibody in the flow-through of the first ion-exchanger, wherein antibody is preferably to be understood as to refer to IgG.
  • the Elisa assay method for validation of these threshold values is described in detail in the experimental section; it should be noted that acidification of the sample to a pH ⁇ 4, preferably in the presence of a mild detergent, is crucial for accurate determination of the amount of leaked protein A.
  • At least 70%, more preferably at least 80%, most preferably at least 90% of the antibody loaded onto the first ion exchanger can be recovered in the flow-through of the ion-exchanger.
  • protein A affinity and subsequent ion exchange chromatography according to the present invention.
  • the method of the present invention is applied to curde, unpurified antibody harvested from serum-free cell culture.
  • the first ion exchanger according to the present invention is an anion exchanger resin; protein A can be bound by both types of resin as described (EP-289 129 B1).
  • the first ion exchanger or anion exchanger can be operated in the column mode at a certain flow rate or in batch operation mode, by submerging the ion exchange resin into the mildly agitated sample solution and further exchanging liquid media by filtration subsequently.
  • the method according to the present invention allows of faster separation of antibody from contaminant protein A.
  • functional groups of such first, anion exchanger that are attached to a matrix support are e.g. primary, secondary, and particularly tertiary or quaternary animo groups such as aminoethyl, diethylaminoethyl, trimethylaminoethyl, trimethylaminomethyl and diethyl-(2-hydroxypropyl)-aminoethyl.
  • Suitable chromatographic support matrixes for the anion exchanger are known in the art.
  • the ion exchanger is a quaternary amine-based anion exchanger mounted on an agarose matrix such as e.g. Sepharose CL-6B or Sepharose Fast Flow (FF) from Amersham-Biosciences/Pharmacia.
  • an agarose matrix such as e.g. Sepharose CL-6B or Sepharose Fast Flow (FF) from Amersham-Biosciences/Pharmacia.
  • FF Sepharose Fast Flow
  • Sepharose QTM from Amersham-Biosciences/Pharmacia.
  • the antibody according to the present invention is a monoclonal antibody that has an isoelectric point (pI) which is at least two pH units above, that is it is more basic than, the pI of the protein A used in the preceding protein A affinity chromatography step; e.g. whereas native protein A has a pI of about 5.0, Streamline recombinant protein A has a pI of about 4.5.
  • the antibody according to the present invention is a monoclonal antibody that has an isoelectric point (pI) which is at least 6.5 or above, more preferably is 7.0 or above, most preferably has an pI of at least 7.5 or above.
  • the pI of the actually harvested and purified antibody refers to the pI of the actually harvested and purified antibody, not the pI that can be simply predicted from the amino acid sequence alone.
  • the actually purified antibody molecule may have undergone further modifications of the polypeptide backbone such as glycosylation, which modifications may add charged moieties and thus may have changed the pI of the molecule.
  • isoelectric focusing Upon determination of pI for product antibody by means of isoelectric focusing (IEF), the microheterogenity of posttranslational processing of the antibody protein, e.g.
  • the ‘pI of an antibody’ refers to that share of antibody product molecules whose pI is within the preferred range of pI as specified above. All further definitions of this description, such as the %-proportion of antibody recovered after a given purification step, refer to said pi-compliant share of antibody only.
  • the pH of buffer used for loading and running the first ion exchanger is set as to put opposing total charge on the antibody and the protein A or protein A contaminant to be separated by means of the ion exchanger in a flow-through mode according to the present invention, taking the pI's of antibody and protein A or protein A derivative into account.
  • this does pertain to average pI value as determinable by experimental means; hence having regard to glycoforms of antibodey, this doesn't mean that a smaller share of glycoforms might be close or be at pI.
  • the mode of operation of a first anion exchanger according to the present invention requires buffer exchange of the acidic or neutralized eluate from the protein A affinity chromatography step with the equilibration buffer of the first anion exchanger.
  • Equilibration buffer and loading buffer are identical in the method of the present invention. Commonly employed ultrafiltration devices such as sold by Amicon or Millipore can be expediently used for that purpose; those avoid the dilution effects whilst using e.g. low molecular weight porous filtration matrices such as Sephadex G-25.
  • the equilibration buffer according to the present invention preferably has a salt concentration of a displacer salt such as e.g.
  • the pH of the equilibration buffer is preferably in the range of pH 6.5 to pH 9.0, more preferably is in the range of pH 7.5 to pH 8.5, most preferably is in the range of pH 7.9 to pH 8.4.
  • N-terminal amino function of a protein has a pKs value of about 9.25, thus binding of contaminant protein A and any other already negatively charged protein to an anion exchanger will get stronger at more basic pH; for a given application, the pH of the loading buffer might need finetuning for optimal discrimination of binding and non-binding for a given pair of antibody and contaminant protein A having differing pI values and different content of cysteine and histidine side chains which may contribute to changes in charge within the selected ranges of pH.
  • a more basic pH interferes with proteinA-antibody interactions as will do any increase in ionic strength; likewise, ionic strength needs finetuning to balance prevention of binding of antibody with the need to abolish binding of contaminant protein A.
  • the ionic strength of the buffer is usually inversely correlated with the pH value; the more strongly protein A gets bound to the anion exchanger depending on pH, the more salt is tolerated for preventing binding of antibody and for interfering with potential proteinA-antibody interactions.
  • the above given ranges for pH and displacer salt thus are to be understood as to be correlated: The lower the pH, the less salt is found permissible within the above given preferred ranges for working the invention.
  • Further salt freight is added by the pH buffering substance, further increasing the ionic strength of the solution.
  • the ionic strength can be determined by measuring the conductivity of the equilibration buffer.
  • the term ‘conductivity’ refers to the ability of an aqueous solution to conduct an electric current between two electrodes measures the total amount of ions further taking charge and ion motility into account. Therefore, with an increasing amount of ions present in the aqueous solution, the solution will have a higher conductivity.
  • the unit of measurement for conductivity is mS/cm (milliSiemens/cm), and can be measured using a commercially available conductivity meter, e.g. from Topac Inc. (Hingham, Mass./U.S.A.) or Honeywell.
  • the loading or equilibration buffer for the first anion exchange step has a conductivity 0.5-5 mS/cm, more preferably of from 1-3 mS/cm, most preferably of from 1.25-2.5 mS/cm. Ideally, it has a conductivity of about 2 mS/cm.
  • suitable buffer salts can be found in Good, N.E. (1986, Biochemistry 5:467-476).
  • E.g. Tris.HCl buffer or a sodium hydrogen phosphate buffer as customarily employed are suitable buffering substances. The concentration of the buffer substance is customarily in the range of e.g.
  • the buffer substance according to the present invention is a phosphate buffer.
  • Hydrogenphosphate has a low elution strength, in particular if employed at a pH at or below pH 8, and further excels by particularly low chaotropic properties.
  • the quaternary, ceramic anion exchanger is a Q-ceramic matrix anion exchanger such as, and particularly preferred, the Q-HyperD® anion exchanger resin sold by Ciphergen Biosystems Ltd., Guildford/Surrey, UK under the ‘Biosepra’ trademark.
  • the above and below mentioned preferred embodiments on pI of antibody, protein load and buffer pH are also preferred in combination with this embodiment, with the exception of the preferred conductivity of buffer when using Q-Hyper D® material being at best 0.5-2 mS/cm, more preferably being in the range of 0.6-1.7 mS/cm, most preferably at about 1 to 1.5 mS/cm and in particular when using Q-Hyper D®-F ion exchanger.
  • This conductivity ensures best purification result in view of deriching contaminant protein A or fragements thereof from the product protein.
  • the Ceramic HYPERD sorbents are made using a rigid porous bead, which is coated and permeated with a functionalized hydrogel.
  • the Ceramic HYPERD sorbents are very easy to use. Their relatively high density makes them easy to pack and use in very large columns. The complete lack of shrinking or swelling eliminates the need for repeated packing/unpacking of columns. Today, columns in excess of 500 liters are used for preparative chromatography of molecules for therapeutic use.
  • the Ceramic HYPERD ion exchangers are also available in a 50 ⁇ m grade (F grade) for preparative processes, with their high capacity and lower back pressure the 50 grade is perfect for capture processes and general downstream processing.
  • the ceramic nature of the bead makes it chemically very stable and it can be cleaned using most commonly used cleaning agents, including 0.5 M NaOH.
  • column operation mode is preferred for the first anion exchanger step.
  • a flow rate of about 10 to 60 ml/h can be advantageously employed.
  • the loading concentration of antibody loaded can favorably be in the range of 10 to 30 mg antibody/ml exchange resin. It goes without saying that the use of extremly diluted samples would give rise to decreased yield of antibody, as is known to the skilled person.
  • the antibody sought to be purified is collected in the low-through of the loading operation including about one column volume of wash with the same equilibration buffer.
  • the pH of the flow-through may be adjusted to neutral pH for improving stability and preventing aggregation and/or precipitation of antibody protein.
  • the antibody is ready for use in applications or may be deemed to require further polishing by customary purification methods.
  • the first ion exchange step is followed by a second ion exchange step in which second step the antibody is loaded and bound by the second ion exchange medium and is eluted with a buffer other than the loading buffer, by means of increased salt and/or pH, as an essentially monomeric, non-aggregated antibody. ‘Essentially’ means less than 5% in this context.
  • the second ion exchanger is a cation exchanger.
  • Such combination of a protein A chromatography step followed by a first anion exchanger and a second cation exchanger step is novel. It is well known that most trace contaminant proteins from cell culture broth have much lower pI values than antibodies, in particular IgG antibodies; cation exchange will therefore allow of efficient removal both of aggregated antibody and potential infectious agents such as virus capsids as well as of protein contaminants other than antibody. Due to speedy operation, highly efficient recovery of antibody after loading, binding to and elution from the co and high capacity of loading, it allows also of repeated, cyclic operation with a single batch of antibody with additive effect of the purification factor achieved in a single round of binding and elution.
  • the pH of the loading buffer is about pH 4 to 7, more preferably pH 4.01 to 6, most preferably pH 4.02 to 5.5.
  • the antibody is eluted from the cation exchanger with a salt gradient in the range of from 0.1 to 1.2 M salt, wherein the salt preferably is an alkaline metal salt, more preferably a lithium, potassium or sodium salt.
  • elution takes place at a pH of from pH 7 to 8 in order to have maximum aggregate removal and minimal damage to antibody due to acidic conditions.
  • the elution takes place at a an acid pH of from pH 4 to 7, more preferably 4.01 to 6 for maximizing removal of contaminant protein A; levels as low as ⁇ 0.4 ng/mg antibody can be realized in this way.
  • This second cation exchanger step renders traditional gel filtration moot whilst allowing of high-capacity as well as fast operation as is typical for ion exchangers. Ion exchangers support loads of 10-30 mg antibody/ml resin.
  • the purification method of a first anion exchanger and a second cation exchanger step in the aftermath of protein A chromatography renders clinical grade antibody in the absence of a further, terminal size exclusion chromatography (SEC) step which SEC step would have a molecular weight cut off suitable for separating antibody aggregates and/or antibody-protein A complexes from monomeric antibody such as an normal IgG.
  • SEC terminal size exclusion chromatography
  • the most appealing feature of the method of the present invention is that purifying antibody via an anion exchanger in a non-binding or flow-through mode, the capacity of the column is not all limiting the through-put of material; the capacity is only decisive with regard to minor amounts of contaminant protein A retain. This saves a lot of processing time and material resources whilst allowing for very efficient removal of protein A contaminant.
  • the detection rabbit antibody was equally purchased from Sigma-Aldrich (#3775). After coating the protein by unspecific adsoprtion process, the coated protein is used to retain protein A-specific protein A capture antibody which capture antibody is further detected with bioinylated rabbit anti-protein A and streptavidin-horseradish peroxidase. Tetramethyl benzidine is used as the chromogenic substrate. Samples of unknown concentration are read off against a standard curve using the very parent-protein A or -protein A derivative of the contaminant protein A sought to be detected. Coating at acidic pH as well as proper preparation of the standard has proven important.
  • Wild-type protein A standard in contrast, is commercially available from a number of companies, e.g. Sigma-Aldrich/Switzerland (#P6031) or Pharmacia (#17-0770-01) and does not require such pretreatment.
  • Sigma-Aldrich/Switzerland #P6031
  • Pharmacia #17-0770-01
  • Preparation of the protein standard was carried out at best immediately prior to use of the standard for coating the microtiter plates.
  • a 1 mg/ml stock solution was prepared and kept at ⁇ 65° C. in a freezer; after thawing, monomeric character of protein A was assayed from an aliquot loaded on non-reducing SDS-PAGE.
  • the concentration of protein standard was determined by Bradford assay (Bradford et al., 1976, Anal. Biochem. 72:248-254; Splittgerber et al., 1989, Anal. Biochem. 179:198-201) as well as by automated amino acid analysis. The result of such pretreatment is shown in FIG.
  • Lane 1 by means of non-reducing 10% SDS-PAGE for a staphylococcal protein A standard (lane 1: native protein A; lane 2: after pretreatment) and pure, uncoupled StreamlineTM recombinant protein A (provided by courtesy of Pharmacia, now Amersham-Biosciences; lane 4: native recombinant protein A; lane 5: after pretreatment).
  • Lane 1 is a molecular weight marker with the corresponding molecular masses being denoted on the vertical axis.
  • every sample is divided into 2 equal volumes of 500 ⁇ l.
  • One is spiked with the 1000 ng/ml spiking solution, or the 10 ⁇ g/ml solution if appropriate, to give a final protein A content of 10 ng protein A per mg of antibody.
  • the other half is spiked with the same volume of sample buffer; thus the dilution factor of the product sample due to spiking is accounted for.
  • Both types of preparation will be referred to as ‘spiked sample’ in the following.
  • the sample buffer was made up from 7.51 g Glycine (base), 5.84 g NaCl, 0.5 ml Triton X-100 to a volume of 1 L with deionized or bidestillated water.
  • the antibody concentrations in the samples were pre-determined by customary Elisa's well known in the art.
  • a further standard solution was spiked with an equal amount of a known standard antibody of comparable constant region affinity for protein A, to determine efficiency of the acidification step and to unravel any potential systematic error introduced by antibody binding to and thus scavenging protein A from capture in the assay.
  • Acidification To 450 ⁇ l of spiked sample or standard is added 200 ul of 0.2 M citrate/0.05% Triton X-100 buffer at pH 3.0. All samples were done in triplicate. Further, dilutions of sample were prepared and tested in triplicate since the assay works optimal for antibody concentrations being in the range of 1 mg/ml and 0.2 mg/ml.
  • the acidification step is crucial in the present assay to liberate contaminant protein A or A fragments which were otherwise bound to the excess of antibody present in the sample solution.
  • Coating buffer was made up from 1.59 g/L Na2CO3, 2.93 g/L NaHCO3 and 0.20 g/L sodium azide. The pH of the buffer was adjusted to pH 9.6. Add 100 ⁇ l antibody solution per well comprising antibody in an amount sufficient as not to show saturation for the protein A standard. Cover plate with plastic film and place in humidity chamber. Incubate at 37° C. overnight for approximately 18 hours.
  • washing buffer [NaCl 5.8 g/L, Na 2 HPO 4 1.15 g/L, NaH 2 PO.H 2 O 0.26 g/L, EDTA 3.7 g/L, Tween-20 0.2 g/L, butanol 10 ml/L, pH 7.2], and tap dry.
  • Add 250 pI blocking buffer [coating buffer with 0.5% casein hammarsten] to each well and incubate for 2 hours at ambient temperature on a benchtop orbital shaker (speed 120 rpm). Rinse all wells three times with at least 300 ⁇ l washing buffer, and tap dry.
  • the substrate solution is prepared like this: A stock solution is prepared by dissolving 10 mg TMB in 1 ml DMSO. 10 ⁇ l of that stock, further 10 ⁇ l of H 2 O 2 are added to a 2.05% (w/w) sodium acetate aequeous solution that was adjusted to pH 6.0 with 0.5 M citric acid. It goes without saying that all water used for preparing any reagent of the assay is of highest quality, that is deionized ultrapure or at least bidestillated water.
  • the substrate solution is incubated at ambient temperature for 8-11 minutes on a shaker.
  • the reaction is then stopped by adding 50 ⁇ l per well of stopping solution [13% H 2 SO 4 ].
  • the absorbance of the wells at wavelength 450 nm is determined on a plate-reading spectrophotometer.
  • the detection limit for such Elisa is 0.2 ng/ml Protein A, with a working range of from 0.2 to 50 ng/ml.
  • the interassay variability is less than 10%.
  • FIG. 2 shows the levels of leaked recombinant protein A in antibody eluates from StreamlineTM recombinant protein A chromatography with single-point attached protein A in thioether linkage.
  • the cycle number refers to repeated use after elution with 1 M sodium chloride and re-equilibration.
  • leakage from cell culture broth from hybridoma cell culture was typically in the order of 500 ppm, other cell types gave levels as high as 1000 ppm.
  • An overview on the rate of leakage from differently sourced matrices is given in Table 1; chromatography was performed according to manufacturer' instruction.
  • FIG. 3 further provides data on insubstantially reduced leakage of contaminant protein A during repeated runs of the protein A affinity chromatography with the same affinity matrix material; wild-type protein A multipoint-attached Sepharose 4 FF (Amersham-Biosciences) was repeatedly used as described in section 2.1 below and the level of contaminant protein A in the eluate, before any further processing of eluate, was determined by Elisa as described above.
  • Cell culture supernatant from a NSO myeloma cell culture was crudely purifed by centrifugation and in depth filtration and concentrated by ultrafiltration; ultrafiltration was also used to exchange the culture fluid to PBS pH 7.5.
  • the titer of the antibody #5 produced by the cells was 0.2 mg/ml, a total of 1 L buffer-exchanged supernatant was loaded.
  • the pI of the monoclonal antibody #5 was 8.5.
  • the protein A StreamlineTM column (5.0 ml volume) was previously equilibrated with 10 column volumes of 50 mM glycine/glycinate pH 8.8, 4.3 M NaCl; flow rate was at 200 cm/h.
  • the column was operated at a flow rate of 50 cmh ⁇ 1 ; loading capacity was about 20 mg/ml matrix material).
  • loading capacity was about 20 mg/ml matrix material.
  • the column was washed with at least 10 column volumes of glycine equilibration buffer supplemented with additional 200 mM NaCl and 0.1% Tween-20. Elution was achieved with elution buffer made up of 0.1 M glycine/HCl pH 4.0 buffer.
  • fractions of eluate comprising the antibody peak were neutralized with an adequate aliquot of 0.5 M Tris HCl pH 7.5 and buffer exchanged with an Amicon diafiltration device with loading/equilibration buffer (10 mM Tris/HCl pH 8.0, 50 mM NaCl) of the present invention for the subsequent anion exchanger step for preventing longer exposure to acidic pH.
  • the antibody concentration and the contaminant protein A concentration were determined as described above.
  • the level of contaminant protein A in the eluate amounted to 1434 ng/mg antibody before and amounted to 1650 ng/mg antibody after diafiltration.
  • the recovery of antibody based on the titer of the buffer exchanged supernatant solution prior to loading was 81%; the concentration of antibody in the diafiltrated solution was 3.6 mg/ml.
  • the purified antibody from section 2.1 was further processed as described: A 5.0 ml Q-Sepharose FF column (Amersham-Biosciences) was packed 10 ml of 0.1 M NaOH, followed by 2 column volumes of 0.1 M Tris pH 8, and equilibrated in 10 column volumes of 10 mM Tris pH 8/50 mM NaCl, at a flow rate of 75 cm/h. After equilibration, the flow rate was reduced to 50 cm/h.
  • the Q-Sepharose column was recycled for further use by separate elution in 2M NaCl and further equilibration as described above.
  • the antibody from exp. 2.2. purified in a non-binding mode by Q-Sepharose anion exchange was used.
  • SP-Sepharose FF allowed of a flow rate of 100 cm/h with a reproducible yield of 93% antibody after loading, washing and elution of the antibody from the cation exchanger.
  • the pH of the antibody solution obtained after Sepharose Q purification step was adjusted to pH 4.5-5.0 with 50 mM acetate buffer pH 4.5.
  • the loading capacity was set with 10 mg/ml matrix material at a conductivity of load of 17 mS/cm.
  • the 50 mM acetate buffer was further used for washing to baseline.
  • a 50 mM Na acetate pH 4.5, 1 M NaCl high salt buffer was used for elution of antibody; monomeric antibody eluted first, whereas aggregates used to elute in the tail fractions at high ionic strength.
  • This multipoint-attached StreamlineTM protein A-affinity matrix was custom made and supplied by Pharmacia Biotech (now Amersham-Pharmacia). It was made up by the manufacturer by coupling the same 34 kD StreamlineTM-type recombinant protein A having a terminal Cys residue to the same Sepharose matrix material, but used traditional CNBr chemistry for activation and coupling instead of epoxide-mediated activation and selective reaction conditions for coupling of —SH groups only (see product information from manufacturer). The method of exp. 2.1 was repeated and the level of contaminant protein A was determined with 353 ng/mg antibody.
  • the Miles patent (U.S. Pat. No. 4,983,722) claims that binding DEAE Sepharose used as a second chromatography step with a salt gradient (0.025M to 0.25M NaCl) for elution can reduce the leached Protein A content in the eluate to less than 15 ng/mg antibody (range of protein A was 0.9 to 14 ng/mg of antibody).
  • 6A1 antibody (pI 6.5-7.5) included two chromatography steps consisting of MabSelect Protein A step followed by Q-Sepharose anion exchange chromatography (non-binding), or DEAE Sepharose chromatography (binding) step . See L0 9007 and L0 9375
  • the culture supernatant containing 6A1 antibody was purified on a MabSelect column (30 ml), connected to an AKTA FPLC system. The conditions used were as described in the table above.
  • the antibody was eluted using 0.1M glycine pH 3.5. Following elution the eluate pH was adjusted to pH 7.0, and then the eluate sample was divided into 5 aliquots; each aliquot was then diafiltered into a different buffer for anion exchange chromatography.
  • the first aliquot was diafiltered into 50 mMTris HCl pH8/75 mMNaCl for Q-Sepharose chromatography run 1.
  • the second aliquot was diafiltered into 50 mMTris HCl pH8/100 mMNaCl for Q-Sepharose chromatography Run 2.
  • the third aliquot was diafiltered into 20 mM sodium phosphate pH6.5/80 mM NaCl for Q-Sepharose chromatography Run 3.
  • Aliquots four and five were buffer exchanged into 25 mMTris HCl pH 8.0/25 mMNaCl for evaluation of binding DEAE Sepharose method described in Miles patent.
  • Run 4 The difference between Runs 4 & 5 is that in Run 4 the main peak was collected as one fraction and diafiltered into standard phosphate buffered saline prior to analysis whereas in Run 5, the elution peak was fractionated and dialysed into a phosphate buffer prepared as described in the Miles patent.
  • DEAE Sepharose Run 4 Column matrix DEAE Sepharose Column dimensions 1.6 cm internal diameter ⁇ 8 cm bed height Column volume 16 mL Column preparation Packed in equilibration buffer at 150 cm/hr Operational flow rate 100 cm/hr (3.35 mL/min) Clean 0.1M Sodium Hydroxide (2 column volumes) Loading capacity 7.5 mg/ml matrix Equilibration 25 mM TrisHCl pH 8.6/25 mM NaCl (8 column volumes) Post load wash 25 mM TrisHCl pH 8.6/25 mM NaCl (5 column volumes) Elution buffer 25 mM TrisHCl pH 8.6/25 mM NaCl To 25 mM TrisHCl pH 8.6/250 mM NaCl (10 column volumes) Wash 2M Sodium Chloride (2 column volumes)
  • a high pI antibody (pI 9.0-9.3) was purified using Protein A Affinity Chromatography (MabSelect—single point attached recombinant Protein A matrix), followed by Q-Sepharose anion exchange chromatography (under non binding conditions; for removal of trace contaminants) followed by SP-Sepharose cation exchange chromatography (under binding conditions for removal of aggregates).
  • Q-HyperD® F Biosepra-Brand of chromatographic supports
  • the processing of a pI 8-9 antibody expressed from NSO cells by Mab-Select Protein A affinity chromatography was conducted essentially as described in example 5. Further essentially as described in example 5 (for Runs 1-3), Q anion exchange chromatography in flow-through mode was then applied to the Protein A-affinity column eluate except that Q-Sepharose, except for a comparative run, was replaced by Q-Hyper DF (Biosepra®) under varying conditions of buffer salt, buffer pH and conductivity. The respective conditions are outlined in the scheme according to FIG.

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US20060194953A1 (en) 2006-08-31
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US7847071B2 (en) 2010-12-07
CN1771260A (zh) 2006-05-10
ES2288252T3 (es) 2008-01-01
KR101200732B1 (ko) 2012-11-13
US20110040075A1 (en) 2011-02-17
DE602004006725T2 (de) 2008-02-07
JP2007525412A (ja) 2007-09-06
ATE363491T1 (de) 2007-06-15
GB0304576D0 (en) 2003-04-02
JP2012067108A (ja) 2012-04-05
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TW200530265A (en) 2005-09-16
TWI325428B (en) 2010-06-01

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