Method for separation of antibodies or antibody fragments being devoid of fc region capable of binding to protein a

Abstract

To address a need for an affinity chromatography separation method of antibodies or antibody fragments which do not have an Fc region capable of binding to a protein A, using a separation resin withstanding alkaline cleaning.SOLUTION: The invention discloses a method for separation of antibodies or antibody fragments, comprising the steps of: a) providing a feed comprising antibodies or antibody fragments having a VH3 region and being devoid of an Fc region capable of binding to a protein A; b) contacting the feed with a separation resin having covalently coupled ligands, wherein the ligands comprise a polypeptide as defined by SEQ ID NO 1 and wherein the antibodies or antibody fragments bind to the separation resin; c) optionally washing the separation resin with washing liquid; d) eluting the antibodies or antibody fragments from the separation resin with elution liquid and recovering the antibodies or antibody fragments.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for the separation of immunoglobulins, and more particularly, for the separation of antibodies or antibody fragments which have a VH3 region and lack an Fc region capable of binding to Protein A. [Background technology]

[0002] Protein A affinity chromatography is widely used for the separation of immunoglobulins, such as therapeutic antibodies. Protein A ligand selectively binds to the Fc region of immunoglobulins, thus allowing a very efficient capture step. For antibody constructs lacking the Fc region, such as antibody fragments, or for immunoglobulins with Fc region variants that do not bind to Protein A, such as IgG3 or IgM, native Protein A may still be useful since it also binds to the VH3 region of immunoglobulins. However, native Protein A is not stable under alkaline cleaning conditions used in bioprocessing, and alkaline-stabilized Protein A variants usually have mutations that inhibit VH3 interactions. See, for example, U.S. Patent Application Publication No. 20060194950, which is incorporated herein by reference in its entirety. This document discusses the inhibition of VH3 interactions by the G29A mutation in the Protein A Fc-binding B domain, which was used in the commercial product MabSelect™ SuRe. Alternative ligands capable of binding antibodies / fragments lacking the Fc region include Protein L, Protein G, and Camelidae antibodies, however, they are all highly sensitive to alkaline cleaning conditions. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Patent Application Publication No. 20060194950 [Patent Document 2] U.S. Patent No. 6,602,990 [Patent Document 3] U.S. Patent No. 7,396,467 [Patent Document 4] U.S. Patent No. 9,802,979 [Patent Document 5] US Patent Application Publication No. 20160288089 [Patent Document 6] International Patent Application No. PCT / EP2019 / 050227 [Patent Document 7] U.S. Patent Application Publication No. 20180094024 [Non-patent literature]

[0004] [Non-Patent Document 1] Altshul et al. (1990) J. Mol. Biol., 215:403~410 [Non-Patent Document 2] S Hjerten: Biochim Biophys Acta 79(2), 393–398 (1964) [Non-Patent Document 3] KR Schmitz et al., Structure 21, 1214-1224, (2013) Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, a need exists for an affinity chromatography separation method for antibodies or antibody fragments without an Fc region that are capable of binding to Protein A, using a separation resin that is resistant to alkaline cleaning. [Means for solving the problem]

[0006] One aspect of the present invention is to provide a method for the isolation of antibodies or antibody fragments, which comprises: a) providing a feed comprising an antibody or antibody fragment having a VH3 region and lacking an Fc region capable of binding to Protein A; b) contacting the feed with a separation resin having a ligand covalently coupled to a support, the ligand being represented by SEQ ID NO:1. AQX 1 AFYEILX 2 LPNLTEEQRX 3 AFIQSLKDDPSVSKAILAEAKKLNX 4 AQ SEQ ID NO:1 (In the sequence, X 1 =E, K, Y, T, F, L, W, I, M, V, A, H, or R, X 2 = H or K, X 3 =N or A, and X 4 =D, F, Y, W, K, or R) wherein said antibody or antibody fragment is bound to said separation resin; c) optionally washing the separation resin with a washing liquid; and d) eluting the antibody or antibody fragment from the separation resin with an elution liquid and recovering the antibody or antibody fragment. This is achieved by a method comprising:

[0007] One advantage is that the method allows the separation of antibodies / antibody fragments that do not contain an Fc region that are capable of binding to Protein A. A further advantage is that the ligand and resin are alkaline stable, surviving repeated cycles of cleaning with up to 2M NaOH.

[0008] Further suitable embodiments of the invention are set forth in the dependent claims. [Brief description of the drawings]

[0009] [Figure 1a] FIG. 1 shows the structure of a typical IgG antibody. [Figure 1b] FIG. 1 shows the structure of a Fab fragment. [Figure 2a]FIG. 1 shows a chromatogram of 10 mg VHH-EgA1 in MabSelect Xtra. [Figure 2b] FIG. 1 shows a chromatogram of 10 mg VHH-EgA1 in MabSelect SuRe. [Figure 2c] FIG. 2 shows a chromatogram of 10 mg VHH-EgA1 in prototype 1. [Diagram 3] FIG. 2 shows a chromatogram of 30 mg / mL VHH-EgA1 in prototype 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] definition The terms "antibody" and "immunoglobulin" are used interchangeably herein and are understood to also include fragments of antibodies, fusion proteins comprising antibodies or antibody fragments, and conjugates comprising antibodies or antibody fragments.

[0011] The terms "Fc binding polypeptide" and "Fc binding protein" refer to a polypeptide or protein, respectively, capable of binding to the crystallizable portion (Fc) of an antibody, and include, for example, Protein A and Protein G, or any fragment or fusion protein thereof which maintains said binding ability.

[0012] The term "antibody or antibody fragment lacking an Fc region capable of binding Protein A" as used herein means either an antibody or antibody fragment lacking an Fc region, or an antibody / antibody fragment having an Fc region that does not bind Protein A. Examples of antibodies / antibody fragments lacking an Fc region include Fab fragments, single chain variable fragments (scFv), domain antibodies, nanobodies, and bi-specific T-cell engagers (BiTe). Examples of antibodies having an Fc region that does not bind Protein A include IgG3 antibodies, IgM antibodies, Camelidae V antibodies, and the like. HExamples of antibodies / fragments that can be used include VH3 single domain antibodies, and any antibody with an Fc region engineered to not bind Protein A. A practical test for whether a particular antibody / fragment has an Fc region capable of binding Protein A is to load it onto a column packed with non-VH3 binding Protein A resin MabSelect™ SuRe (GE Healthcare) in a loading buffer of 20 mM phosphate, 500 mM NaCl, pH 7, wash the column first with the loading buffer, then with a wash buffer of 50 mM citrate, pH 6, and elute the column with an elution buffer of 50 mM citrate, pH 2.5. If the antibody / fragment does not have an Fc region capable of binding Protein A, it will be found mainly in the loading flow-through and / or the wash buffer. If it has such an Fc region, it will be found mainly in the eluate.

[0013] The term "linker" as used herein refers to an element that connects two polypeptide units, monomers, or domains to each other in a multimer.

[0014] The term "spacer" as used herein refers to an element that connects a polypeptide or polypeptide multimer to a support.

[0015] The term "% identity" in the context of amino acid sequence comparisons is determined by standard alignment algorithms such as, for example, the Basic Local Alignment Tool (BLAST™) described in Altshul et al. (1990) J. Mol. Biol., 215:403-410. Web-based software for this is freely available from the US National Library of Medicine at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome. Here, the algorithm "blastp (protein-protein BLAST)" is used to align a query sequence with a subject sequence and, inter alia, determine its % identity. The term "native protein A" as used herein refers to a polypeptide comprising five native immunoglobulin binding domains E, D, A, B, C directly linked to each other as defined by SEQ ID NO: 31. The polypeptide may further comprise a leader sequence at the N-terminus and a coupling element at the C-terminus or N-terminus.

[0016] As used herein, the terms "comprises," "comprising," "containing," "having," and the like, may have the meaning ascribed to them in United States patent law and may mean "includes," "including," and the like; similarly, "consisting essentially of" or "consists essentially" have the meaning ascribed to them in United States patent law, and the terms are open-ended, allowing for the presence of more than what is recited, but excluding prior art embodiments, so long as the presence of more than what is recited does not change its basic or novel characteristics that are recited.

[0017] In one aspect, the present invention discloses a method for the isolation of antibodies or antibody fragments without an Fc region capable of binding to Staphylococcus Protein A. The method comprises the steps of: a) Providing a feed, e.g. a clarified cell culture supernatant, comprising antibodies or antibody fragments having a VH3 region and lacking an Fc region capable of binding to Protein A. In particular, the target species in the feed to be separated can be antibody fragments having a VH3 region and lacking an Fc region. They can be for example selected from the group consisting of Fab, scFv, domain antibodies, nanobodies and BiTe. The target species in the feed to be separated can also be antibodies having a VH3 region and an Fc region that does not bind to Protein A. They can be for example IgG3, IgM and Camelidae V. H The antibody or antibody fragment may be selected from the group consisting of VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15, VH16, VH17, VH18, VH19, VH20, VH21, VH22, VH23, VH24, VH25, VH36, VH37, VH38, VH39, VH40, VH41, VH42, VH43, VH44, VH45, VH46, VH47, VH48, VH49, VH50, VH51, VH52, VH53, VH54, VH55, VH56, VH57, VH58, VH60, VH61, VH62, VH63, VH64, VH65, VH66, VH67, VH68, VH69, VH70, VH71, VH72, VH73, VH74, VH75, VH75, VH76, VH77, VH78, VH79, VH80, VH81, VH82, VH83, VH84, VH85, VH86, VH87, VH88, VH89, VH90, VH91, VH92, VH93, VH94, VH95, VH96, VH97, VH98, VH99, VH100, VH101, VH102, VH103, VH104, VH105, VH106, VH107, VH108, VH109, ​​VH110, VH11

[0018] b) contacting the feed with a separation resin having a ligand covalently coupled to a support, the ligand being represented by SEQ ID NO:1 AQX 1 AFYEILX 2 LPNLTEEQRX 3 AFIQSLKDDPSVSKAILAEAKKLNX 4 AQ SEQ ID NO:1 (In the sequence, X 1 =E, K, Y, T, F, L, W, I, M, V, A, H, or R, X 2 = H or K, X 3 =N or A, and X4 =D, F, Y, W, K, or R) or a polypeptide having at least 85% identity, such as at least 90% or at least 95% identity to SEQ ID NO: 1, and said antibody or antibody fragment is bound to said separation resin. The binding strength of the antibody or antibody fragment to the separation resin may be in the nanomolar range or stronger.

[0019] c) optionally washing the separation resin with a washing liquid to remove contaminants and / or impurities. The washing liquid may for example be a buffer of pH 5-7.

[0020] d) eluting said antibody or antibody fragment from said separation resin with an elution liquid and recovering said antibody or antibody fragment. The elution liquid may suitably be a buffer of pH 2-5.

[0021] After step d), the method may further comprise step e) of cleaning said separation resin with a cleaning liquid having a pH of 13 or higher. The cleaning liquid may comprise 0.1-2M, such as 0.5-2M, of an alkali metal hydroxide, such as NaOH.

[0022] In some embodiments, steps a) through e) are repeated at least 50 times, such as at least 200 times.

[0023] In certain embodiments, X 1 =E,X 2 =H,X 3 =N and / or X 4 =D. Examples of such embodiments include: SEQ ID NO:2 AQEAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKAILEAAKLNDAQ; SEQ ID NO:3 AQKAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKAILEAAKLNDAQ; SEQ ID NO:4 AQEAFYEILKLPNLTEEQRNAFIQSLKDDPSSVSKAILAEAKKLNDAQ; SEQ ID NO:5 AQEAFYEILHLPNLTEEQRAAFIQSLKDDPSVSKAILAEAKKLNDAQ; and SEQ ID NO:6 AQEAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKAILAEAKKLNWAQ

[0024] In some embodiments, the ligand comprises a multimer of a polypeptide P. Such multimers may suitably be linked by a linker region L comprising 0-25 amino acid residues, such as 0-15 amino acid residues, e.g., the structure (PL) n-1 -P or L-(PL) n-1-P. The multimer may be, for example, a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, or nonamer. It may be a homomultimer, where all units in the multimer are identical, or it may be a heteromultimer, where at least one unit is different from the others. Advantageously, all units in the multimer are alkaline stable, for example by including a mutation as disclosed above. The polypeptides may be linked to each other directly by a peptide bond between the C-terminus and the N-terminus of the polypeptide. Alternatively, two or more units in the multimer may be linked by a linker comprising an oligomeric or polymeric species, for example a peptide having up to 25 or 30 amino acid residues, for example 3-25, 3-20, or 3-15 amino acid residues. The linker may comprise or essentially consist of an amino acid sequence defined by or having at least 80%, at least 90%, or at least 95% identity thereto, for example selected from the group consisting of APKVDAKFDKE, APKVDNKFNKE, APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE, APKKFDKE, APK, APKYEDGVDAKFDKE, and YEDG, or alternatively selected from the group consisting of APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE, APKKFDKE, APKYEDGVDAKFDKE, and YEDG. They may also be defined by, or consist essentially of, an amino acid sequence selected from the group consisting of APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE, APKKFDKE, APK, and APKYEDGVDAKFDKE, or a peptide sequence having at least 80%, at least 90%, or at least 95% identity thereto.

[0025] The nature of such a linker should preferably not destabilize the spatial conformation of the protein unit. This can be achieved, for example, by avoiding the presence of proline in the linker. Furthermore, the linker should also preferably be stable enough in an alkaline environment so as not to impair the properties of the mutant protein unit. For this purpose, it is advantageous if the linker does not contain asparagine. It may be even more advantageous if the linker does not contain glutamine. The multimer may further comprise a number of amino acid residues at the N-terminus, for example constituting residues from a signal sequence resulting from or cleaved off from the cloning process. The number of additional amino acid residues may be 20 or less, for example 15 or less, for example 10 or less, or 5 or less. As a particular example, the multimer may comprise AQ, AQGT, VDAKFDKE, AQVDAKFDKE, or AQGTVDAKFDKE (also called leader sequence) at the N-terminus. The multimer may, for example, comprise or consist essentially of a sequence as defined by SEQ ID NO:7, or having at least 80%, at least 90%, or at least 95% identity thereto. SEQ ID NO:7 AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC

[0026] In some embodiments, the polypeptides and / or multimers as disclosed above further comprise one or more coupling elements at the C-terminus or N-terminus selected from the group consisting of one or more cysteine ​​residues, multiple lysine residues, and multiple histidine residues. The coupling element may also be located within 1-5 amino acid residues, such as 1-3 or 1-2 amino acid residues, from the C-terminus or N-terminus. The coupling element may for example be a single cysteine ​​at the C-terminus. The coupling element may be directly linked to the C-terminus or N-terminus or it / they may be linked via a stretch comprising up to 15 amino acids, such as 1-5, 1-10, or 5-10 amino acids. This stretch should also preferably be sufficiently stable in an alkaline environment so as not to impair the properties of the mutant protein. For this purpose, it is advantageous if the stretch does not contain asparagine. It may be even more advantageous if the stretch does not contain glutamine. The advantage of having a C-terminal cysteine ​​is that end-point coupling of proteins can be achieved through reaction of the cysteine ​​thiol with an electrophilic group on the support. This provides excellent mobility of the coupled protein, which is important for binding capacity.

[0027] The polypeptide or multimer can be attached to the support by conventional coupling techniques utilizing, for example, thiol groups (in cysteine), amino groups (in lysine or at the N-terminus), and / or carboxyl groups (in aspartic acid or glutamic acid or at the C-terminus) present in its ligand. Bisepoxides, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS), etc. are well-known coupling reagents. Molecules known as spacers can be introduced between the support and the polypeptide / multimer, which improve the accessibility of the polypeptide / multimer and facilitate the chemical coupling of the polypeptide / multimer to the support. Depending on the nature of the polypeptide / multimer and the coupling conditions, the coupling can be a multi-point coupling (e.g., via multiple lysines) or a single-point coupling (e.g., via a single cysteine).

[0028] In certain embodiments, the polypeptide or multimer is coupled to the support via a thioether bond. Methods for performing such coupling are well known in the art and are easily performed by those skilled in the art using standard techniques and equipment. Thioether bonds are flexible and stable, and are generally suitable for use in affinity chromatography. Specifically, when the thioether bond is via a terminal or near-terminal cysteine ​​residue on the polypeptide or multimer, the mobility of the coupled polypeptide / multimer is enhanced, which provides improved binding capacity and binding reaction rate. In some embodiments, the polypeptide / multimer is coupled via a provided C-terminal cysteine ​​on the protein, as described above. This allows for efficient coupling of the cysteine ​​thiol with electrophilic groups on the support, such as epoxide groups, halohydrin groups, etc., resulting in a thioether cross-link coupling.

[0029] In certain embodiments, the support comprises a polyhydroxypolymer, such as a polysaccharide. Examples of polysaccharides include, for example, dextran, starch, cellulose, pullulan, agar, agarose, etc. Polysaccharides are hydrophilic in nature, have a low degree of non-specific interactions, they offer a high content of reactive (activatable) hydroxyl groups, and they are generally stable to alkaline cleaning solutions used in bioprocessing.

[0030] In some embodiments, the support comprises agar or agarose. The support used in the present invention can be easily prepared according to standard methods such as reversed-phase suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964)). Alternatively, the base matrix is ​​a commercially available product such as cross-linked agarose beads sold under the name SEPHAROSE™ FF (GE Healthcare). In certain embodiments, which are particularly advantageous for large-scale separations, the support is adapted to increase its rigidity using the methods described in U.S. Pat. Nos. 6,602,990 or 7,396,467 (incorporated herein by reference in their entirety), thus making the matrix more suitable for high flow rates.

[0031] In certain embodiments, supports such as polymer, polysaccharide, or agarose supports are crosslinked, such as with hydroxyalkyl ether crosslinks. Crosslinking reagents that produce such crosslinks can be epihalohydrins such as epichlorohydrin, diepoxides such as butanediol diglycidyl ether, allylation reagents such as allyl halides or allyl glycidyl ether. Crosslinking is beneficial to the rigidity of the support and improves its chemical stability. Hydroxyalkyl ether crosslinks are alkaline stable and do not cause significant nonspecific adsorption.

[0032] In some embodiments, the solid support is in the form of a filter (e.g., a membrane or depth filter matrix). In particular, the support may comprise one or more sheets or membranes of cellulose nanofibers, as described, for example, in U.S. Pat. Nos. 9,802,979, 20160288089, and International Patent Application No. PCT / EP2019 / 050227, which are incorporated herein by reference in their entirety. The cellulose nanofibers may suitably be crosslinked for improved chemical and mechanical stability. EXAMPLES

[0033] Example 1 Single heavy chain variable domain (V H H) expression Single heavy chain variable domain (V) antibodies from camelid origin, also known as nanobodies H H) was heterologously expressed in Escherichia coli (E. coli). H The H fragment is the sequence SEQ ID NO:23 from KR Schmitz et al., Structure 21, 1214-1224, (2013): >4KRN:A|PDBID|CHAIN|SEQUENCE QVQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYTYYTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSLEHHHHHH Based on.

[0034] This sequence was modified for periplasmic expression by use of the OmpA signal peptide (E. coli, outer membrane protein A, UniProt P0A910) and a signal peptide cleavage site was introduced as a dipeptide AQ followed by an enzyme restriction site for KpnI (amino acids VD). The final protein after processing of the signal peptide had the sequence found in SEQ ID NO:24 below: AQVDQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYTYYTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSLEHHHHHH

[0035] This sequence was generated by DNA synthesis company ATUM (CA, USA) with codon optimization from a proprietary E. coli expression vector and cloned into an expression vector (plasmid) containing an IPTG-inducible promoter. The plasmid was transformed into chemically competent E. coli K12 cells. The plasmid was dissolved in 25 mM Tris, pH 8.5 at a concentration of 20 ng / μl. 1 μl of the plasmid was added to 50 μl of thawed competent cells on ice bath and cooled for 20 minutes. Then, the cells were heat shocked at 42°C for 60 seconds, followed by cooling on ice bath for 2 minutes. After cooling the cells on ice, 950 μl of Luria Broth (LB) was added and the cells were incubated for 60 minutes at 225 rpm with shaking. After incubation, the cells were plated on Luria Agar (LA) plates with 100 μl on each plate and the plates were incubated overnight in an oven set at 37°C. The following day, a single colony was picked and grown in LB to a final OD 600nm of 1, then supplemented with 15% glycerol and frozen at -80°C until further use. The frozen cell bank was thawed and 100 μl was used to inoculate 100mL Terrific Broth (TB) supplemented with 50mg / L kanamycin and grown overnight at 37°C with shaking. The following day, 10mL of the overnight culture was used to inoculate 750mL fermentation medium supplemented with 50mg / L kanamycin. The culture was induced with a final concentration of 1mM IPTG when it reached an optical density (OD) of 80 measured at 600nm. The culture was maintained at constant temperature and pH for 12 hours after induction under aeration and agitation. After 24 hours, the fermentation was terminated and the cells were separated from the supernatant by centrifugation at 2500xg for 30 minutes. The supernatant was discarded and the cells were resuspended in 600 mL PBS and heat-incubated at 48° C. for 3 hours to release periplasmically expressed proteins. The heat-treated suspension was centrifuged at 10,000×g for 30 minutes and subsequently filter-sterilized using a 0.2 μm filter.The clarified supernatant was loaded onto a HiScale 16 column (GE Healthcare, Uppsala, Sweden) packed with 24 mL MabSelect™ resin (GE Healthcare, Uppsala, Sweden) equilibrated with 20 mM phosphate, pH 7, 500 mM NaCl. The column was washed with 50 mM citrate, pH 6 to reduce the conductivity in the elution pool, followed by elution with 50 mM citrate, pH 2.5. Purified V. H The H fragment had a concentration of 7.7 g / L in a pool volume of 11 mL. The elution pool was adjusted to pH 7 by the addition of 2 M tris base.

[0036] Example 2 V for different chromatography resins H VH3 binding of H antibody fragment The VHH fragments generated in Example 1 were tested for binding to different chromatography resins, MabSelect™ Xtra (GE Healthcare, Uppsala, Sweden), MabSelect™ SuRe (GE Healthcare, Uppsala, Sweden) and Prototype 1 (highly cross-linked agarose beads with a median bead diameter (d50,v) of 57 μm coupled with a hexameric ligand of SEQ ID NO: 7 by the method described in US Patent Publication No. 20180094024, which is incorporated herein by reference in its entirety). All experiments were performed using an AKTA Pure 150 FPLC system (GE Healthcare, Uppsala, Sweden). MabSelect™ Xtra and MabSelect™ SuRe were tested using pre-packed 1 mL HiTrap™ columns (GE Healthcare, Uppsala, Sweden). Prototype 1 was packed into a Tricorn 5 column (GE Healthcare, Uppsala, Sweden) with 1.94 mL column volume (CV). All column chromatography runs were performed with 20 mM phosphate, 500 mM NaCl, pH 7 as running buffer, the second with a buffer consisting of 50 mM citrate, pH 6, and 50 mM citrate, pH 2.5 as elution buffer. To test binding to different resins, 10 mg V of the 1000 ng / ml IgG prepared in Example 1 was used. H The H fragment was diluted in 7 mL running buffer (20 mM phosphate, pH 7, 500 mM NaCl) and loaded onto a 1 mL Mabselect™ Xtra, SuRe, or Prototype 1 column. Results from the chromatography run are shown in Figures 2a)-c). V bound to the MabSelect™ Xtra column H The H fragment was recovered at a rate of 73%, and V bound to prototype 1. HThe H fragment had a recovery of 86%. However, the fragment bound very weakly to the MabSelect™ SuRe resin with only a 4% recovery, with the majority of the protein found in either the loading or washing steps. Furthermore, the dynamic binding capacity for prototype 1 was H H fragments were measured by loading to 10% breakthrough using a 6 minute residence time. A Tricorn 5 / 100 column was packed with prototype 1 to 1.98 mL CV. The capacity was measured using 20 mM phosphate, 150 mM NaCl, pH 7.4 as running buffer and 50 mM acetate, pH 3.5 as elution buffer B. The column was regenerated with 0.5 M NaOH between runs. H H fragment was diluted to 2.3 mg / mL in 50 mL running buffer and filtered through a Sterivex 0.22 μm filter. The final concentration was determined by UV measurement at 280 nm. The column was equilibrated at a flow rate of 1 mL / min before starting the run. The maximum absorbance of the sample was measured by running the sample in the bypass. Sample loading was performed at a flow rate of 0.33 mL / min (6 min residence time) until the absorbance at 280 nm reached 10% of the absorbance maximum. A wash step was performed at a flow rate of 1 mL / min for 10 CV, followed by an isocratic elution with buffer B. CIP was performed with 0.5 M NaOH for 3 CV at a flow rate of 1 mL / min. Peaks greater than 100 mAU were collected at the selected outlet. The dynamic binding capacity for prototype 1 resin was determined at 10% breakthrough (Q ) using a 6 min residence time. B10 ) was 36 mg / mL.

[0037] Figure 2a) Column: MabSelect™ Xtra Sample: 10 mg VHH-EgA1 (SEQ ID NO: 25) Buffer: Starting: 20 mM phosphate, 500 mM NaCl, pH 7 Wash 2: 50 mM citrate, pH 6 Eluent: 50mM citrate, pH 2.5 Figure 2b) Column: MabSelect™ SuRe Sample: 10 mg VHH-EgA1 (SEQ ID NO:25) Buffer: Starting: 20 mM phosphate, 500 mM NaCl, pH 7 Wash 2: 50 mM citrate, pH 6 Eluent: 50mM citrate, pH 2.5 Figure 2c) Column: Prototype 1 Sample: 10 mg VHH-EgA1 (SEQ ID NO: 25) Buffer: Starting: 20 mM phosphate, 500 mM NaCl, pH 7 Wash 2: 50 mM citrate, pH 6 Eluent: 50mM citrate, pH 2.5

[0038] Example 3 Fab VH3 binding VH3-type Fabs were generated from full-length monoclonal antibodies (mAbs) by cleavage with papain. The mAb solution (54 ml, 1836 mg) was adjusted to pH 7.5 by addition of 2 M Tris base and then diluted 1:1 in digestion buffer (25 mM Na phosphate, 1 mM EDTA, 5 mM mercaptoethanol, pH 7.5). Papain crystals were added to the solution (21 mg). The solution was incubated overnight at 37°C. The following day, 400 μl of antipain (papain inhibitor) was added to the digested mAb. The solution was loaded onto a HiScale 26 / 135 Capto L column (GE Healthcare, Uppsala, Sweden) to purify the cleaved Fab fragments. The digested pool was loaded onto a Capto L column equilibrated with 25 mM Tris, pH 8, followed by a second wash with 50 mM Na-citrate, pH 5.0, and elution with 50 mM Na-citrate, pH 2.3. The Fab was analyzed by SEC using a Superdex™ 200 Increase (GE Healthcare, Uppsala, Sweden) to verify complete cleavage of the mAb. The dynamic binding capacity for prototype 1 was measured by loading the Fab to 10% breakthrough using a 6 min residence time. A Tricorn 5 / 100 column was loaded with prototype 1 to 1.98 mL CV. The capacity measurements were performed using 20 mM phosphate, 150 mM NaCl pH 7.4 as running buffer and 50 mM acetate, pH 3.5 as elution buffer B. The column was regenerated with 0.5 M NaOH between runs. Fab was diluted to 2.25 mg / mL in 100 mL running buffer and filtered through a Sterivex 0.22 μm filter. The final concentration was determined by UV reading at 280 nm. The column was equilibrated at a flow rate of 1 mL / min before starting the run. The maximum absorbance of the sample was measured by running the sample in the bypass. Sample loading was performed at a flow rate of 0.33 mL / min (6 min residence time) until the absorbance at 280 nm reached 10% of the absorbance maximum.A wash step was performed at a flow rate of 1 mL / min for 10 CV, followed by an isocratic elution with Buffer B. CIP was performed with 0.5 M NaOH at a flow rate of 1 mL / min for 3 CV. Peaks greater than 100 mAU were collected at the selected outlet. The dynamic binding capacity for Prototype 1 resin was 106 mg / mL at 10% breakthrough (QB10) using a 6 min residence time.

[0039] Example 4 Single heavy chain variable domain (V H H) Four point mutations in the fragment sequence Protein A and a single heavy chain variable domain (V H H) To test the robustness of the interaction between the fragments, a series of point mutations were made and subsequently tested for retention of affinity to MabSelect™ Xtra, SuRe, and Prototype 1. HThe H sequence was generated by DNA synthesis company ATUM (CA, USA) with a proprietary codon optimization for E. coli expression and cloned into an expression vector (plasmid) containing an IPTG-inducible promoter. The plasmid was transformed into chemically competent E. coli K12 cells. The plasmid was dissolved in 25 mM Tris, pH 8.5 at a concentration of 20 ng / μl. 1 μl of the plasmid was added to 50 μl of thawed competent cells on ice bath and cooled for 20 minutes. Then, a heat shock was performed at 42°C for 60 seconds, followed by cooling on ice bath for 2 minutes. After cooling the cells on ice, 950 μl of Luria Broth (LB) was added and the cells were incubated for 60 minutes at 225 rpm with shaking. After incubation, the cells were plated on Luria Agar (LA) plates with 100 μl on each plate and the plates were incubated overnight in an oven set at 37°C. The following day, a single colony was picked and grown in LB to a final OD 600nm of 1, then supplemented with 15% glycerol and frozen at -80°C until further use. The frozen cell bank was thawed and 500μl was used to inoculate 100mL Terrific Broth (TB) supplemented with 50mg / L kanamycin. The culture was grown at 37°C until an OD 600nm of 1 was reached, then induced with IPTG at a final concentration of 1mM. After induction, the culture was grown overnight at 37°C and stopped the following morning by centrifugation at 2300xg for 20 minutes. The pellet was resuspended in 25mM Tris-HCl, 500mM NaCl, pH 8.0, then sonicated (40% amplitude, 2 minutes, 5 seconds on / 3 seconds off), centrifuged at 10000xg for 10 minutes, and filtered through a 0.2μm filter. The samples were then loaded onto MabSelect™ Xtra, Mabselect™ SuRe, and Prototype 1 columns to verify binding. Flow-through fractions were collected until an elution peak was observed.If no peak was obtained, the flow-through was loaded onto a Ni-Sepharose FF column (GE Healthcare, Uppsala, Sweden) using 25 mM Tris pH 8.0 as running buffer and a 5 CV gradient of 50 mM Tris, 250 mM imidazole, pH 8.0 as elution buffer.

[0040] [Table 1]

[0041] V H H-EgA1 (T57K), SEQ ID NO:25 AQVDQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYKYYTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSLEHHHHHH V H H-EgA1 (T57K, K64R), SEQ ID NO:26 AQVDQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYKYYTDSVRGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSLEHHHHHH V H H-EgA1 (T57P), SEQ ID NO:27 AQVDQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYPYYTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSLEHHHHHH V H H-EgA1 (T57S), SEQ ID NO:28 AQVDQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYSYYTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSLEHHHHHH V H H-EgA1 (T57R), SEQ ID NO:29 AQVDQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYRYYTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSLEHHHHHH V H H-EgA1 (S70T), SEQ ID NO: 30 AQVDQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYTYYTDSVKGRFTITRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSLEHHHHHH

[0042] This written description uses examples to disclose the invention, including the best mode, and also enables any person skilled in the art to practice the invention, including making and using any device or system and practicing 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 words of the claims, or if they include structural elements that are equivalent to the literal words of the claims with insignificant differences. All patents and patent applications mentioned herein are incorporated by reference in their entirety, as if each was individually incorporated.

Claims

1. 1. A method for the isolation of an antibody or an antibody fragment comprising: a) providing a feed comprising an antibody or antibody fragment having a VH3 region and lacking an Fc region capable of binding to Protein A; b) contacting the feed with a separation resin having a ligand covalently coupled to a support, the ligand being represented by SEQ ID NO:

1. AQX 1 AFYEILX 2 LPNLTEEQRX 3 AFIQSLKDDPSVSKAILAEAKKLNX 4 AQ SEQ ID NO:1 (In the sequence, X 1 = E, K, Y, T, F, L, W, I, M, V, A, H, or R, X 2 = H or K, X 3 =N or A, and X 4 =D, F, Y, W, K, or R) wherein said antibody or antibody fragment is bound to said separation resin; c) optionally washing the separation resin with a washing liquid; and d) eluting the antibody or antibody fragment from the separation resin with an elution liquid and recovering the antibody or antibody fragment. A method comprising:

2. X 1 2. The method of claim 1, wherein:

3. X 2 3. The method of claim 1 or 2, wherein: =K.

4. X 3 4. The method of claim 1, wherein =N.

5. X 4 5. The method of claim 1, wherein:

6. 6. The method of any one of claims 1 to 5, wherein the ligand comprises multimers of the polypeptides linked by a linker region comprising from 0 to 15 amino acid residues.

7. The method of claim 6, wherein the multimer is a tetramer, pentamer, or hexamer.

8. 8. The method of claim 6 or 7, wherein the multimer is coupled to the support via a thioether bond derived from a C-terminal cysteine ​​on the multimer.

9. 9. The method of any one of claims 1 to 8, wherein the support comprises cross-linked agarose beads.

10. 10. The method of claim 1, wherein the support comprises crosslinked cellulose nanofibers.

11. 11. The method of any one of claims 1 to 10, wherein the feed is a clarified cell culture supernatant.

12. The method of any one of claims 1 to 11, wherein the antibody or antibody fragment is an antibody fragment having a VH3 region and lacking an Fc region.

13. 13. The method of any one of claims 1 to 12, wherein the antibody fragment is selected from the group consisting of a Fab, an scFv, a domain antibody, a nanobody, and a BiTe.

14. 14. The method according to any one of claims 1 to 13, wherein the binding strength of the antibody fragment to the separation resin is in the nanomolar range or stronger.

15. 15. The method of any one of claims 1 to 14, wherein the antibody or antibody fragment is capable of binding to native Protein A via the VH3 region.

16. 16. The method according to any one of claims 1 to 15, wherein the method comprises a step c) in which contaminants and / or impurities are removed.

17. The method of claim 16, wherein the washing liquid is a buffer of pH 5-7.

18. The method according to any one of claims 1 to 17, wherein the elution liquid is a buffer of pH 2 to 5.

19. 19. The method according to any one of claims 1 to 18, further comprising after step d) a step e) of cleaning the separation resin with a cleaning liquid having a pH of 13 or higher.

20. The method of claim 19, wherein the cleaning liquid comprises 0.5 to 2M alkali metal hydroxide.

21. 21. The method of any one of claims 1 to 20, wherein steps a) to e) are repeated at least 50 times.

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