US20120071637A1 - Selective enrichment of antibodies - Google Patents

Selective enrichment of antibodies Download PDF

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US20120071637A1
US20120071637A1 US13/040,361 US201113040361A US2012071637A1 US 20120071637 A1 US20120071637 A1 US 20120071637A1 US 201113040361 A US201113040361 A US 201113040361A US 2012071637 A1 US2012071637 A1 US 2012071637A1
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protein
binding
seq
affinity
precipitation
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Dorothee Ambrosius
Michael DIETERLE
Philine DOBBERTHIEN
Maria-Katharina HOYER
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Boehringer Ingelheim International GmbH
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Boehringer Ingelheim International GmbH
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Assigned to BOEHRINGER INGELHEIM INTERNATIONAL GMBH reassignment BOEHRINGER INGELHEIM INTERNATIONAL GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DIETERLE, MICHAEL, AMBROSIUS, DOROTHEE, DOBBERTHIEN, PHILINE, HOYER, MARIA-KATHARINA
Priority to US14/151,949 priority Critical patent/US10894806B2/en
Priority to US17/123,656 priority patent/US20210107937A1/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
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/32—Extraction; Separation; Purification by precipitation as complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Definitions

  • the present invention relates to purification processes for proteins, particularly immunoglobulins or other proteins that have an Fc domain.
  • Biomolecules such as proteins, polynucleotides, polysaccharides and the like are increasingly gaining commercial importance as medicines, as diagnostic agents, as additives in foods, detergents and the like, as research reagents and for many other applications.
  • the need for such biomolecules can no longer normally be met—for example in the case of proteins—by isolating molecules from natural sources, but requires the use of biotechnological production methods.
  • the biotechnological preparation of proteins typically begins with the cloning of a DNA fragment into a suitable expression vector. After transfection of the expression vector into suitable prokaryotic or eukaryotic expression cells and subsequent selection of transfected cells the latter are cultivated in bioreactors and the desired protein is expressed. Then the cells or the culture supernatant is or are harvested and the protein contained therein is worked up and purified.
  • the separation of impurities is of major importance, in addition to the product yield.
  • the process-dependent impurities contain components of the host cells such as proteins (host cell proteins, HCP) and nucleic acids and originate from the cell culture (such as components of the medium) or from the working up (such as for example salts or dissolved chromatography ligands).
  • Product-dependent impurities are molecular variants of the product with different properties. These include shortened forms such as precursors and hydrolytic breakdown products, but also modified forms, produced for example by deaminations, incorrect glycosylations or wrongly linked disulphide bridges.
  • polymers and aggregates are also included among the product-dependent variants.
  • Other impurities are contaminants. This term covers all other materials of a chemical, biochemical or microbiological nature that do not belong directly to the production process. Examples of contaminants include viruses that may occur in undesirable manner in cell cultures.
  • Affinity chromatography matrices are used as the stationary phase in the industrial purification of various substances. Immobilised ligands can be used for specifically concentrating and purifying substances that have a certain affinity for the particular ligand used.
  • immobilised protein A is often used as the initial purification step. Protein A is a protein with about 41 kDa of Staphylococcus aureus that binds with high affinity (10 ⁇ 8 M-10 ⁇ 12 M of human IgG) to the CH 2 /CH 3 domain of the Fc region of immunoglobulins.
  • immunoglobulins or fusion proteins that have a protein A-binding Fc region from the mobile phase bind specifically to the protein A ligand, which is covalently coupled to a carrier (e.g. Sepharose).
  • a carrier e.g. Sepharose
  • Protein A from Staphylococcus aureus (wild-type protein A) and genetically modified recombinant protein A (rec. protein A) interacts through non-covalent interactions with the constant region (Fc fragment) of the antibodies. This specific interaction can be used to separate impurities efficiently from the antibody.
  • the interaction between the antibody and the protein A ligand can be deliberately stopped and the antibody released or eluted from the stationary phase.
  • Protein A Apart from protein A as affinity ligand, there are many other molecules currently known that bind to the Fc fragment. Thus, individual domains of protein A are used instead of the complete protein (8). Protein variants are known, which differ precisely from the B domain of the protein A, which are suitable for binding Fc-fragment-containing molecules (16, 17). These different variants differ essentially in the mutations that have been inserted in order to increase the stability or binding affinity. These mutants of the B domain are usually known as Z-domain or protein Z. Besides protein A or protein G, various peptides are also suitable for selective binding to the Fc fragment (14). The present great interest in affinity ligands for the Fc fragment lead one to suppose that still more affinity ligands are being found.
  • Affinity chromatography and particularly the frequently used protein A chromatography are expensive, however, and precisely when there are increasing product concentrations in the fermenter and large quantities of product, there are limits on the chromatographic purification processes that can be carried out.
  • the critical points are: loading capacities, number of cycles, process times, pool volumes and quantities of buffer. In the future, therefore, alternative purification processes will be essential.
  • a general overview of conventional purification strategies, including affinity chromatography and alternative methods of affinity chromatography, can be found in the following articles (7+10).
  • a more recent method of affinity chromatography uses not a constantly immobilised affinity ligand but, to begin with, a solubilised affinity ligand that is mixed with the target protein (11).
  • the affinity ligand carries a fusion tag or a fusion protein which makes it possible to carry out the immobilisation of the affinity ligand on a solid phase that takes place in the second step.
  • the target protein is separated from the ligand under suitable conditions and thus eluted from the column.
  • the invention described here uses affinity precipitation instead of affinity chromatography to purify biomolecules. This method appears to have great potential precisely when used on a larger scale (1, 2).
  • Affinity precipitation is the most effective method of protein precipitation (2).
  • the precipitation of a protein from a solution in general is a well known process that is frequently used.
  • many proteins have already been separated from a solution by ammonium sulphate precipitation from a protein mixture.
  • macromolecules e.g. proteins
  • this step leads to precipitation.
  • this precipitation usually takes place non-specifically.
  • affinity precipitation makes use of the selective binding of an affinity molecule to a target molecule.
  • Affinity molecules may be for example proteins, peptides, oligonucleotides or small chemical molecules.
  • affinity precipitation between two principles, first and second order affinity precipitation (7).
  • first order affinity precipitation both the affinity molecule and the target molecule have two binding sites, so that it is possible for a network to form between the two molecules and an affinity complex is formed that sediments at a specific size.
  • affinity macroligands AML are used, for example.
  • the affinity molecule is bound to a stimulatable substance, usually a polymer.
  • the stimulatable substance changes its solubility characteristics as a result of a change in the ambient conditions, such as e.g. a shift in the pH or temperature, and precipitation occurs.
  • a bifunctional affinity molecule or for a bifunctional target molecule either.
  • affinity ligands are at present coupled to polymers or other mediators (15).
  • Smart polymers or stimuli-responsive “intelligent” polymers or affinity macroligands
  • These stimuli may be for example changes in pH or temperature (12-13).
  • smart polymers react to their stimuli with precipitation in solution. This precipitation can be reversed, after the desired separation of the supernatant solution, by suitable conditions.
  • Smart polymers can be conjugated with various biomolecules, leading to a large accumulation of polymer/biomolecule systems that can be used for all kinds of applications. Examples of these biomolecules include proteins, oligonucleotides and sugars.
  • affinity precipitation is the recently described “affinity sinking” method.
  • affinity precipitation a linking molecular scaffold is used to bind a number of affinity ligands to one another. This then makes it possible to form the network required for precipitation. The binding of the affinity ligand to the network former may take place both non-covalently and covalently.
  • This method was recently described in the patent “Compositions and methods for purifying and crystallizing molecules of interest” (6). In this, first of all a solution containing antibodies is mixed with an affinity ligand that is covalently bound to a crosslinker. No precipitation is observed. Only after the subsequent addition of a coordinating ion or molecule does precipitation take place. In a second similar application the affinity ligand is linked to a biotin-binding protein which forms a network with the mediator avidin (9).
  • U.S. Pat. No. 7,083,943 describes an affinity precipitation in which a binding domain for the target protein is linked to a scaffold domain the amino acid sequence of which is intended to assist the tendency to precipitation.
  • the present invention relates to affinity precipitation using a binding protein with two binding sites, which dispenses completely with an additional fusion protein or a linker molecule.
  • the affinity protein on its own is sufficient for the precipitation, and there is no need for a stationary phase.
  • the invention used here also makes it easier to recover the affinity protein.
  • the invention relates in particular to a process for the selective concentration of immunoglobulins or other proteins that contain an Fc domain (target protein), comprising the following steps:
  • the invention relates to a process in which the Fc-binding protein is a dimer of an Fc-binding domain of protein A or protein G.
  • the two monomers of the dimer are linked to one another via a disulphide bridge.
  • the invention relates to a process in which the dimer is a homodimer, the monomers of which have the SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or a sequence that differs from SEQ ID NO. 1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.
  • the Fc-binding protein is used in a ratio of 0.5-20 in relation to the target protein.
  • the solution in step a. preferably has a pH of 5.5-8.
  • the undoing of the binding in step d. takes place at a pH of 2-4.5.
  • the invention relates to an Fc-binding protein consisting of 2 identical sub-units that have the sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a sequence that differs from SEQ ID NO. 1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, the two sub-units being linked to each other via a covalent bond.
  • the covalent bond is a disulphide bond.
  • FIG. 1 SDS-PAGE of the Z dimer. To demonstrate the dimerisation via cysteines, iodoacetamide or iodoacetamide with dithiothreitol was added before the application to the SDS-PAGE.
  • FIG. 2 Affinity precipitation using Z-dimer.
  • protein Z is oxidised.
  • Z-dimer is added to a solution containing antibody (mAB), which leads to the selective precipitation of the antibody.
  • FIG. 3 UV diagram of ion exchange chromatography under acid conditions for separating the Z dimer from the antibody (Experiment 2).
  • the UV chromatogram at 220 nm is shown in red
  • the UV chromatogram at 280 nm is shown in blue
  • the increasing content of buffer B (20 mM phosphate, 3 M NaCl) is shown in green.
  • FIG. 4 SDS-PAGE for Experiment 2.
  • the present invention relates to methods of depleting impurities, particularly host cell protein (HCP) and DNA, from protein compositions of the kind obtained from cell cultures in which proteins are expressed recombinantly or endogenously.
  • the invention relates to methods of purifying or concentrating a protein (target protein) by binding an Fc-binding protein or multimer thereof with at least two binding sites. In further steps the precipitate is separated off and then the binding of the Fc-binding protein to the target protein is removed using suitable conditions.
  • the present invention relates to a process for the selective concentration of immunoglobulins or other proteins that contain an Fc domain (target protein), comprising the following steps:
  • the target protein may be in particular an immunoglobulin or a protein that contains the Fc domain of an immunoglobulin and can bind to protein A or fragments of protein A.
  • Immunoglobulins consist of two heavy and two light chains.
  • the heavy chains each have one variable and three to four constant domains depending on the immunoglobulin. These are referred to analogously as VH and CH1, CH2, CH3.
  • the variable domains of a light and a heavy chain form the antigen binding site.
  • the domain CH2 contains a carbohydrate chain which forms a binding site for the complement system.
  • the CH3 domain contains the Fc-receptor binding site.
  • Target proteins to which the process according to the invention can be applied are all proteins that have an Fc domain. Examples of proteins that contain CH2/CH3 regions are antibodies, immunoadhesins and fusion proteins in which the protein of interest is connected to a CH2/CH3 region.
  • the target protein is for example an antibody that has a CH2/CH3 region and is thus capable of binding to protein A.
  • CH2/CH3 region refers to the amino acids in the Fc region of an antibody that interact with protein A.
  • the Fc-binding protein comprises according to the invention precisely two binding sites for one Fc domain.
  • the invention relates to a process wherein the Fc-binding protein is a dimer of an Fc-binding domain of protein A or protein G.
  • the two monomers of the dimer are preferably linked together by a disulphide bridge.
  • Fc-binding protein proteins or peptides which are capable of binding to the Fc region.
  • Fc-binding proteins bind with a dissociation constant (K D value) in the range from 10 ⁇ 2 -10 ⁇ 13 M.
  • the Fc-binding protein is a homo- or heterodimer of Fc-binding domains which comprise or contain the sequences listed in Table 1:
  • a homodimer is meant, in this context, an Fc-binding protein that is made up of two sub-units of the same sequence.
  • heterodimer an Fc-binding protein that is made up of two sub-units of different sequences, each of which has a binding site for an Fc domain.
  • the sub-units contain sequences that are selected from the sequences in Table 1.
  • the invention relates to a process in which the dimer is a homodimer the monomers of which have SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or a sequence that differs from SEQ ID NO. 1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.
  • the monomers that differ from SEQ ID NO. 1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids have the property of binding Fc-domains with a K D value in the range from 10 ⁇ 2 -10 ⁇ 13 M.
  • Binding conditions are conditions in which binding to the target protein by the Fc-binding proteins takes place, preferably in a pH range of pH 5.5-9, preferably 6-8.
  • the precipitation occurs spontaneously under binding conditions, such as those found for example in cell-free eukaryotic culture supernatant.
  • polymers e.g. polyethyleneglycols
  • the Fc-binding protein is used in a molar ratio of 0.5-20 relative to the target protein.
  • the separation of the precipitate may be carried out by centrifugation and subsequent removal of the supernatant, but also by filtration techniques.
  • the undoing of the binding to the target protein is carried out under conditions that enable the Fc-binding protein to be separated from the target protein.
  • this can be done by adjusting the pH to a range between pH 2 and 4.5.
  • the invention in another aspect relates to an Fc-binding protein that consists of 2 identical sub-units that have the sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a sequence that differs from SEQ ID NO. 1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, wherein the two sub-units are linked together by a covalent bond.
  • the covalent bond is a disulphide bond.
  • Protein Z was obtained as a recombinant protein from E. coli .
  • the removal of impurities was carried out after separation of the cell debris by ion exchange chromatography.
  • the cell culture supernatant of CHO cells optimised to secretory production was obtained by filtration or centrifugation after several days' culture.
  • the ion exchange chromatography was carried out on an ⁇ KTA Explorer 10 apparatus (GE Healthcare) with observation of the UV absorption at 220 and 280 nm.
  • the column material used was SP Sepharose FF (19 mL gel bed volume). After the application of the pellet resuspended at an acid pH, the column was equilibrated with 20 mM phosphate buffer at pH 3.3 and then the antibody and the Z-dimer were separated from one another over a 25 column volume long gradient. A 20 mM phosphate buffer with 3 M NaCl at pH 3.3 was used for the elution.
  • Protein A HPLC on a Waters or Dionex apparatus (injector pumps and column oven W2790/5, UV detector W2489) was used to determine the antibody content of cell culture-free supernatant and purified antibody.
  • the antibody content of the solutions was determined using the UV signal of the acidically eluting peak.
  • a 20% homogeneous SDS gel was used to test the individual fractions and supernatants.
  • the protein bands were detected by silver staining according to Heukeshoven.
  • the DNA analysis was carried out after single strand production by an enzymatically catalysed detecting reaction.
  • a 50 kDa Centramate Sheet PES (polyethersulphone) made by Messrs Pall with a surface area of 180 cm 2 was used for the UF/DF tests in Experiment 3.
  • the UF/DF was carried out so that the antibody was initially concentrated six-fold and then diafiltered (exchanged for 6 volumes of the buffer) before being circulated for 10 min with the retentate valve fully open. The diafiltration was repeated twice.
  • the total UF/DF was carried out with 50 mM acetate buffer+100 mM arginine+150 mM NaCl pH 3.0.
  • the hydrophobic interaction chromatography (Experiment 3) was carried out with a 27 mL column on an ⁇ KTA apparatus.
  • the column material used was Toyopearl Phenyl 650 M made by Tosoh.
  • 3.5 M ammonium sulphate buffer was added to the retentate until a conductivity of 165 mS/cm was obtained.
  • Detection of the dimerisation of protein Z by cysteine was carried out by SDS-PAGE analysis ( FIG. 1 ). To ensure that the working up does not induce any oxidation of the protein Z, first of all the free cysteine groups were alkylated with iodoacetamide. For detecting the monomer, additionally reduction was carried out with dithiothreitol (DTT). 2.6 nmol of Z-dimer were mixed with DTT or a corresponding volume of buffer and incubated for five minutes at 95° C. Then the mixture was cooled to ambient temperature and iodoacetamide or a corresponding volume of buffer was added and the mixture was incubated for a further 20 min in the dark. After five minutes' incubation with SDS-PAGE buffer the preparation was applied to the SDS-PAGE.
  • DTT dithiothreitol
  • the SDS-PAGE analysis shows that the Z monomer is formed by the addition of the reducing agent DTT and the gel band of the Z dimer disappears.
  • the tests described below show that the selective precipitation of an antibody can be achieved with a Z-dimer ( FIG. 2 , Experiment 1).
  • the Z-dimer is the dimerised B-domain of protein A from Staphylococcus aureus linked via two non-native cysteines, which carries a mutation compared with the wild-type (cf. Equipment and Methods).
  • Experiment 2 demonstrates that the pellet obtained can be put back into solution by resuspension in the acid pH range and then an antibody can be removed again from the Z-dimer by ion exchange chromatography at an acid pH.
  • the mixture was centrifuged for 10 min at 4000 rpm and the supernatant was separated from the pellet.
  • the antibody content of the supernatant was determined by means of the UV signal of analytical SEC and by protein A chromatography (Equipment and Methods).
  • the content of antibody in the pellet was determined by subtracting the antibody content of the supernatant from the total content of antibody used. A precipitation of up to 99% antibody was observed (test 1: 99%/test 2: 76%).
  • the culture supernatant was examined for protein impurities before and after the precipitation by analytical SEC.
  • the chromatogram from the cell-free eukaryotic culture supernatant was compared directly with the supernatant after precipitation.
  • Protein impurities may be both host cell proteins and fragments or aggregates of the target protein.
  • a cation exchanger (SP Sepharose FF) was used to separate the Z-dimer from the antibody. 0.2 ⁇ mol of purified antibody were incubated with 0.2 ⁇ mol of Z-dimer in a volume of 10.1 mL for 36 min. After subsequent centrifugation (4000 rpm, 10 min) the supernatant was removed. Then the pellet was dissolved batchwise in 10 mL phosphate buffer at pH 7.4 and centrifuged again (4000 rpm, 10 min). The pellet thus obtained was resuspended in 20 mL phosphate buffer (20 mM phosphate, pH 3.3) and purified using an ion exchanger. The Z-dimer could be separated from the antibody through a gradient over 25 bed volumes ( FIGS. 3 and 4 ).
  • a multi-step antibody purification process was carried out with affinity precipitation as the capture step.
  • the total yield of the antibody was 98% after the precipitation and decreased to 87% after UF/DF and to 64% after HIC.
  • DNA and host cell protein analysis showed that the DNA could be reduced by 99% by the precipitation step followed by UF/DF and the host cell protein content could be reduced by 99.9% compared with the initial value (cell-free culture supernatant).

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