Patent Application
20220362744
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 7, 2019, is named 34428_0389_ST.txt and is 5,750 bytes in size.
The present invention relates to separation matrices, and more particularly to a separation matrix useful in antibody separation. The invention also relates to a method of separating antibodies on the matrix.
In the manufacturing of therapeutic monoclonal antibodies (mAbs), affinity chromatography on matrices comprising coupled Staphylococcus Protein A (SpA) or variants of SpA is commonly used as a first separation step to remove most of the contaminants. As the demand for therapeutic mAbs is increasing there is a strong driving force for improving the efficiencies of the separation processes and several approaches are under evaluation.
Multicolumn continuous chromatography processes are available, where the feed is applied to a first column and is then diverted to one or more subsequent columns as the first columns approaches saturation and the first column is eluted and regenerated to be loaded again during elution and regeneration of the subsequent column(s). Such processes can be denoted periodic countercurrent chromatography (PCC) or simulated moving bed (SMB) and are of considerable interest for separation of therapeutic mAbs, see e.g. U.S. Pat. No. 7,901,581, US20130248451, US20130280788 and U.S. Pat. No. 7,220,356, which are hereby incorporated by reference in their entireties. PCC/SMB processes can significantly increase the productivity, but it appears that the full potential cannot be reached with currently available separation matrices, which are designed for conventional batch chromatography.
Accordingly, there is a need for new separation matrices specifically designed for continuous chromatography processes and for processes using such matrices. There is also a general need for separation matrices providing high dynamic binding capacities at very short residence times, in particular when used in shallow beds with low hydraulic resistance.
One aspect of the invention is to provide a separation matrix allowing separation of mAbs in shallow beds with high productivity. This is achieved with a separation matrix comprising porous particles to which antibody-binding protein ligands have been covalently immobilized, wherein the density of said ligands is above 5 mg/ml and the volume-weighted median diameter of said porous particles is at least 10 and below 30 μm.
One advantage is that the matrix has a high binding capacity at very short residence times.
A second aspect of the invention is to provide a chromatography column allowing continuous separation of mAbs with high productivity. This is achieved with a column as defined in the claims.
A third aspect of the invention is to provide a multicolumn chromatography system allowing continuous separation of mAbs with high productivity. This is achieved with a system as defined in the claims.
A fourth aspect of the invention is to provide an efficient method of separating antibodies. This is achieved with a method as defined in the claims. One advantage is that the method allows very short residence times with high binding capacity.
Further suitable embodiments of the invention are described in the dependent claims.
The terms “antibody” and “immunoglobulin” are used interchangeably herein, and are understood to include also fragments of antibodies, fusion proteins comprising antibodies or antibody fragments and conjugates comprising antibodies or antibody fragments.
The terms an “Fc-binding polypeptide” and “Fc-binding protein” mean a polypeptide or protein respectively, capable of binding to the crystallisable part (Fc) of an antibody and includes e.g. Protein A and Protein G, or any fragment or fusion protein thereof that has maintained said binding property.
The term “linker” herein means an element linking two polypeptide units, monomers or domains to each other in a multimer.
The term “spacer” herein means an element connecting a polypeptide or a polypeptide multimer to a support.
The term “% identity” with respect to comparisons of amino acid sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST™) described in Altshul et al. (1990) J. Mol. Biol., 215: 403-410. A web-based software for this is freely available from the US National Library of Medicine (blast.ncbi.nlm.nih.gov). Here, the algorithm “blastp (protein-protein BLAST)” is used for alignment of a query sequence with a subject sequence and determining i.a. the % identity.
As used herein, the terms “comprises,” “comprising,” “containing,” “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
In one aspect, illustrated by
The porous particles may comprise a crosslinked polysaccharide, which provides a large hydrophilic surface for coupling of the ligands, with minimal risk of non-specific interactions between mAbs or contaminants and the particles. The polysaccharide suitably has zero or very low (e.g. <5 micromol/ml) content of charged groups to prevent non-specific interactions. The crosslinking increases rigidity and chemical stability and can be achieved by methods known in the art, in particular by epoxide crosslinking, using e.g. epichlorohydrin or a diepoxide as crosslinker. Examples of polysaccharides can be dextran, cellulose and agarose. Agarose has the particular advantage that highly porous, rigid gels can be achieved by thermal gelation of aqueous agarose solution. The agarose can suitably be crosslinked by the methods described in U.S. Pat. No. 6,602,990, 7,396,467 or 8,309,709, which are hereby incorporated by reference in their entireties. Agarose crosslinked by these methods, so called high flow agarose, has a particularly advantageous combination of high rigidity and high porosity/pore volume, allowing high flow rates and rapid mass transport. High rigidity is particularly important for matrices having small particle sizes, to allow high flow rates without collapse of the matrix. The agarose can e.g. be allylated with reagents like allyl glycidyl ether or allyl halides before gelation, as described in U.S. Pat. No. 6,602,990. To allow for high binding capacities and rapid mass transport, the particles can advantageously have a large volume of pores accessible to macromolecular species like IgG antibodies. This can be determined by inverse size exclusion chromatography (SEC) as described in “Handbook of Process Chromatography, A Guide to Optimization, Scale-Up and Validation” (1997) Academic Press, San Diego, Gail Sofer & Lars Hagel eds. ISBN 0-12-654266-X, p. 368. A suitable parameter for the accessible pore volume is the gel phase distribution coefficient, KD, determined for a probe molecule of defined size. This is a column-independent variable calculated from the retention volume VR for the probe molecule, the interstitial void volume of the column V0 and the total liquid volume of the column Vt according to KD=(VR−V0)/(Vt−V0). The porous particles can suitably have a KD value in the range of 0.5-0.9, such as 0.6-0.85 or 0.65-0.85, for dextran of molecular weight 110 kDa as the probe molecule.
The ligands can e.g. be derived from antibody-binding bacterial proteins or derivatives thereof, such as SpA (Protein A), Peptostreptococcus Protein L or Streptococcus Protein G. Alternatively they can e.g. be derived from antibodies, such as single-chain camelid antibodies. They may bind to antibodies such that the affinity constant for the interaction is at most 1×10−6M, for example at most 1×10−7M, such as at most 1×10−9M. They can comprise an Fc-binding protein, such as SpA or and SpA variant, which binds to the Fc part of IgG molecules. They can comprise monomers, dimers or multimers of native or mutated Protein A Fc-binding domains. The native Protein A Fc-binding domains E, D, A, B and C are shown in
The ligands may comprise monomers, dimers or multimers of Protein A domains, typically wherein one or more of said domains have been mutated. One or more of the domains may e.g. be derived from Protein Z or the B or C domain of Protein A with the amino acid residue at position 23 being a threonine.
The ligands may additionally comprise one or more linker sequences of 1-10 amino acid residues, e.g. VDNKFN, ADNKFN, VDAKFD, AD or FN, suitably between the individual domains. In addition, the ligands may comprise a coupling moiety, e.g. a cysteine or a plurality of lysines at the C-terminus or N-terminus of the ligand, suitably at the C-terminus. The ligands may also comprise a leader sequence at the N-terminus, e.g. a scar or a residue after cleavage of a signal peptide and optionally also a copy of a linker sequence. Such a leader sequence may e.g. be a 1-15 amino acid (e.g. a 1-10 amino acid) peptide, e.g. AQ, AQGT, AQVDAKFD, AQGTVDAKFD or AQVDNKFN. Hence, a typical structure of a ligand may e.g. be Leader—(Domain-Linker)n-1—Domain—Coupling moiety. n may e.g. be 1-7, such as 1, 2, 3, 4, 5, 6 or 7.
In a second aspect, illustrated by
In a third aspect, illustrated by
In a fourth aspect, the invention discloses a method of separation of antibodies by affinity chromatography. This method comprises the steps of:
The method can suitably be carried out in the chromatography system 10 disclosed above. It can typically be carried out as a capture step, following clarification of a cell culture supernatant feed comprising the antibodies. After step d), the recovered eluent with antibodies may be subjected to further chromatography steps, such as ion exchange, multimodal chromatography and/or hydrophobic interaction chromatography steps.
In certain embodiments of the method, in step a) an effluent from the first chromatography column 11 is passed through a second chromatography column 12 packed with the same separation matrix as the first column;
The residence time in steps a), a′) and a″) may e.g. be less than 2 min, such as 0.3-1 min or 0.3-0.8 min.
The method may further, after steps c), c′) and c″) respectively, comprise steps e), e′) and e″), each comprising conveying a cleaning liquid through said first, second and third chromatography columns respectively. The cleaning liquid can be an aqueous alkali solution comprising at least 0.1M (e.g. 0.1-1M or 0.1-0.5 M) alkali. 0.5-1 M alkali solutions can also be used as sanitizing solutions. The alkali may e.g. be NaOH, but can also be e.g. KOH. The cleaning (also called cleaning in place—CIP) step ensures that any residual feed components are removed from the columns before repetition of the binding and elution steps. Suitably, the ligands are capable of withstanding repeated alkali treatments, e.g. as discussed above where the matrix retains at least 95% of its original IgG-binding capacity after 5 h incubation with 0.5 M NaOH.
After steps e), e′) and e″) respectively, the method may also comprise equilibration steps f), f′) and f″) to reequlibrate the columns for steps a), a′) and a″) respectively.
The base matrices used were a set of rigid cross-linked agarose bead sieve fraction samples of 17-50 micrometers (volume-weighted, d50V) median diameter (determined on a Malvern Mastersizer 2000 laser diffraction instrument), prepared according to the methods of U.S. Pat. No. 6,602,990 and with a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.50-0.82 for dextran of Mw 110 kDa, according to the methods described in “Handbook of Process Chromatography, A Guide to Optimization, Scale-Up and Validation” (1997) Academic Press, San Diego, Gail Sofer & Lars Hagel eds. ISBN 0-12-654266-X, p. 368, using HR10/30 columns (GE Healthcare) packed with the prototypes in 0.2 M NaCl and with a range of dextran fractions as probe molecules (flow rate 0.2 ml/min). The base matrix prototypes were sieved to obtain desired particle size distributions.
100 ml base matrix was washed with 10 gel volumes distilled water on a glass filter. The gel was weighed (1 g=1 ml) and mixed with 30 ml distilled water and 8.08 g NaOH (0.202 mol) in a 250 ml flask with an agitator. The temperature was adjusted to 27+/−2° C. in a water bath. 16 ml epichlorohydrin (0.202 mol) was added under vigorous agitation (about 250 rpm) during 90+/−10 minutes. The reaction was allowed to continue for another 80+/−10 minutes and the gel was then washed with >10 gel volumes distilled water on a glass filter until neutral pH was reached. This activated gel was used directly for coupling as below.
To 16.4 mL of ligand solution (50 mg/mL) in a 50 ml Falcon tube, 139 mg NaHCO3, 17.4 mg Na2CO3, 143.8 mg NaCl and 141 mg EDTA, was added. The Falcon tube was placed on a roller table for 5-10 min, and then 63 mg of DTE was added. Reduction proceeded for >45 min. The ligand solution was then desalted on a PD10 column packed with Sephadex G-25. The ligand content in the desalted solution was determined by measuring the 276 nm UV absorption.
The activated gel was washed with 3-5 GV {0.1 M phosphate/1 mM EDTA pH 8.6} and the ligand was then coupled according to the method described in U.S. Pat. No. 6,399,750 5.2.2, although with considerably higher ligand amounts (see below). All buffers used in the experiments had been degassed by nitrogen gas for at least 5-10 min. The ligand content of the gels was controlled by varying the amount and concentration of the ligand solution, adding 5-20 mg ligand per ml gel. The ligand was a tetramer of SEQ ID NO:8, with a VDAKFD linker and a C-terminal cysteine, except in prototypes 902A and B where it was a hexamer of SEQ ID NO:10 with a VDAKFD linker and a C-terminal cysteine.
After immobilization the gels were washed 3×GV (gel volumes) with distilled water. The gels+1 GV 10.1 M phosphate/1 mM EDTA/10% thioglycerol pH 8.61 was mixed and the tubes were left in a shaking table at room temperature overnight. The gels were then washed alternately with 3×GV 10.1 M TRIS/0.15 M NaCl pH 8.61 and 0.5 M HAc and then 8-10×GV with distilled water. Gel samples were sent to an external laboratory for amino acid analysis and the ligand content (mg/ml gel) was calculated from the total amino acid content. Table 1 shows the ligand contents of the coupled prototypes and the Kd and d50v data for the corresponding base matrices.
The Qb10% dynamic capacity for polyclonal human IgG at 0.5, 1, 2.4 and 6 mM residence time was determined as outlined below.
Gammanorm 165 mg/ml (Octapharma), diluted to 2 mg/ml in Equilibration buffer.
PBS Phosphate buffer 20 mM+0.15 M NaCl, pH 7.4
PBS Phosphate buffer 20 mM+0.15 M NaCl, pH 7.4
100 mM acetate pH 2.9
0.5 or 1 ml of resin was packed in TRICORN™ 5/50 columns (bed height 2.5-5 cm). The breakthrough capacity was determined with an ÄKTAExplorer 10 system at residence times of 0.5-6 minutes, with flow rates adjusted according to the residence time desired for the resin volume in question. Equilibration buffer was run through the bypass column until a stable baseline was obtained. This was done prior to auto zeroing. Sample was applied to the column until a 100% UV signal was obtained. Then, equilibration buffer was applied again until a stable baseline was obtained.
Sample was loaded onto the column until a UV signal of 85% of maximum absorbance was reached. The column was then washed with 5 column volumes (CV) equilibration buffer at flow rate 0.5 ml/min. The protein was eluted with 5 CV elution buffer at a flow rate of 0.5 ml/min. Then the column was cleaned with 0.5M NaOH at flow rate 0.2 ml/min and re-equilibrated with equilibration buffer.
For calculation of breakthrough capacity at 10% (q10%), the equation below was used. q10% is the amount of IgG that is loaded onto the column until the concentration of IgG in the column effluent is 10% of the IgG concentration in the feed.
The dynamic binding capacity (DBC) at 10% breakthrough was calculated. The dynamic binding capacity (DBC) was calculated for 10 and 80% breakthrough.
This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims. Any patents or patent applications mentioned in the text are hereby incorporated by reference in their entireties, as if they were individually incorporated.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1703116.2 | Feb 2017 | GB | national |
This application is a divisional of and claims the priority benefit of U.S. application Ser. No. 16/486,653, filed Aug. 16, 2019, which claims the priority benefit of PCT/EP2018/053816, filed on Feb. 15, 2018, which claims priority benefit of Great Britain Application No. 1703116.2 filed on Feb. 27, 2017, the entire contents of which are hereby incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16486653 | Aug 2019 | US |
| Child | 17853514 | US |
