Method for the isolation of an antibody or antibody fragment lacking an Fc region capable of binding to protein A

Abstract

The present invention discloses a method for the separation of antibodies or antibody fragments, comprising: a) providing a feed containing antibodies or antibody fragments 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 covalently coupled ligand, wherein the ligand comprises a polypeptide as defined by SEQ ID NO: 1, such that the antibodies or antibody fragments bind to the separation resin; c) optionally washing the separation resin with a wash liquid; d) eluting the antibodies or antibody fragments from the separation resin with an elution liquid and recovering the antibodies or antibody fragments.

Description

Technical Field

[0001] The present invention relates to the separation of immunoglobulins, and more specifically, to a method for separating antibodies or antibody fragments that have a VH3 region and lack an Fc region capable of binding to protein A.

Background Art

[0002] Protein A affinity chromatography is widely used for the separation of immunoglobulins such as therapeutic antibodies. The protein A ligand selectively binds to the Fc region of immunoglobulins, thus enabling a very efficient capture step. For antibody constructs lacking an Fc region, such as antibody fragments, or for immunoglobulins having an Fc region variant that does not bind to protein A, such as IgG3 or IgM, native protein A can still be useful because it also binds to the VH3 region of the immunoglobulin. However, native protein A is not stable under the alkaline cleaning conditions used in bioprocessing, and alkali-stabilized protein A variants usually have mutations that inhibit VH3 interactions. See, for example, U.S. Patent Application Publication No. 20060194950, which is hereby incorporated 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 used in the commercially available product MabSelect(™) SuRe. Alternative ligands capable of binding to antibodies / fragments lacking an Fc region include protein L, protein G, and camelid antibodies. However, they are all very sensitive to alkaline cleaning conditions.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

[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, there is a need 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, said antibody or antibody fragment having the ability to bind to Protein A; b) contacting the feed with a separation resin having a ligand coupled by a covalent bond to a support, wherein the ligand is SEQ ID NO: 1 AQX1AFYEILX2LPNLTEEQRX3AFIQSLKDDPSVSKAILAEAKKLNX4AQSEQ ID NO: 1 (in the sequence, X1 = E, K, Y, T, F, L, W, I, M, V, A, H, or R, X2 = H or K, X3 = N or A, and X4 = D, F, Y, W, K, or R) comprising a polypeptide as defined by, and the antibody or antibody fragment binding to the 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 is achieved by a method comprising.

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

[0008] Further suitable embodiments of the invention are described in the dependent claims.

Brief Description of the Drawings

[0009]

Fig. 1a

Fig. 1b

Fig. 2a

Fig. 2b

Fig. 2c

Fig. 3

Mode for Carrying Out the Invention

[0010] Definition The terms “antibody” and “immunoglobulin” are used interchangeably herein and are understood to include antibody fragments, fusion proteins containing an antibody or antibody fragment, and conjugates containing an antibody or antibody fragment.

[0011] The terms “Fc-binding polypeptide” and “Fc-binding protein” mean a polypeptide or protein, respectively, that has the ability to bind to the crystallizable part (Fc) of an antibody, and include, for example, protein A and protein G, or any fragment or fusion protein thereof that maintains said binding ability.

[0012] The term “antibody or antibody fragment lacking an Fc region capable of binding to 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 to protein A. Examples of antibodies / antibody fragments lacking an Fc region include Fab fragments, single-chain variable fragments (scFv), domain antibodies, nanobodies, and bispecific T-cell engagers (BiTe). Examples of antibodies having an Fc region that does not bind to protein A include IgG3 antibodies, IgM antibodies, camelid V HExamples include H single domain antibodies and any antibody having an Fc region engineered not to 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 and 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 flow-through fraction and / or the wash buffer. If it has such an Fc region, it will be found mainly in the eluate.

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

[0014] As used herein, the term "spacer" means an element that connects a polypeptide or polypeptide multimer to a support.

[0015] The term "% identity" with respect to the comparison of amino acid sequences is determined by standard alignment algorithms such as the Basic Local Alignment Tool (BLAST (trademark)) 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 target sequence and, in particular, to determine its % identity. As used herein, the term "native protein A" means a polypeptide comprising five native immunoglobulin-binding domains E, D, A, B, and 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 other like terms may have the meaning ascribed to them in US patent law and may mean "includes", "including", and other like terms; similarly, "consisting essentially of" or "consists essentially" has the meaning ascribed in US patent law, and the term is open-ended and allows for more than the recited elements as long as the basic or novel features of the recited one are not changed by the presence of more than the recited ones, but excludes prior art embodiments.

[0017] In one aspect, the present invention discloses a method for separating an antibody or antibody fragment that does not have an Fc region capable of binding to protein A of Staphylococcus. The method includes the following steps: a) Preparing a feed containing an antibody or antibody fragment that has a VH3 region and lacks an Fc region capable of binding to protein A, for example, a clarified cell culture supernatant. Specifically, the target species in the feed to be separated can be an antibody fragment that has a VH3 region and lacks an Fc region. They can be selected, for example, from the group consisting of Fab, scFv, domain antibodies, nanobodies, and BiTe. The target species in the feed to be separated can also be an antibody that has a VH3 region and an Fc region that does not bind to protein A. They can be selected, for example, from the group consisting of IgG3, IgM, and camelid V H H single domain antibodies. Overall, the antibody or antibody fragment can have the ability to bind to native protein A via the VH3 region. For non-Fc antibodies / fragments, this can be easily tested by checking whether they bind to MabSelect™ or MabSelect Xtra resin (GE Healthcare) containing a native protein A ligand coupled by covalent bond as defined above.

[0018] b) Contacting the feed with a separation resin having a ligand coupled to a support by covalent bond, wherein the ligand has the sequence of SEQ ID NO: 1 AQX1AFYEILX2LPNLTEEQRX3AFIQSLKDDPSVSKAILAEAKKLNX4AQ SEQ ID NO: 1 (In the sequence, X1 = E, K, Y, T, F, L, W, I, M, V, A, H, or R, X2 = H or K, X3 = N or A, and X4 = D, F, Y, W, K, or R) A step of including a polypeptide as defined by [[ID=]], or a polypeptide having at least 85% identity, such as at least 90% or at least 95% identity with SEQ ID NO: 1, and the antibody or antibody fragment binds to the separation resin. The binding strength between the antibody or antibody fragment and the separation resin can be within the nanomolar range or stronger.

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

[0020] d) A step of eluting the antibody or antibody fragment from the separation resin with an elution liquid and recovering the antibody or antibody fragment. The elution liquid can suitably be a buffer with a pH of 2 to 5.

[0021] After step d), the method can further include step e) of cleaning the separation resin with a cleaning liquid having a pH of 13 or higher. The cleaning liquid can contain 0.1 to 2 M, for example 0.5 to 2 M, of an alkali metal hydroxide, such as NaOH.

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

[0023] In certain embodiments, X1 = E, X2 = H, X3 = N, and / or X4 = D. Examples of such embodiments include the following: SEQ ID NO: 2 AQEAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKAILAEAKKLNDAQ; SEQ ID NO: 3 AQKAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKAILAEAKKLNDAQ; SEQ ID NO: 4 AQEAFYEILKLPNLTEEQRNAFIQSLKDDPSVSKAILAEAKKLNDAQ; SEQ ID NO: 5 AQEAFYEILHLPNLTEEQRAAFIQSLKDDPSVSKAILAEAKKLNDAQ; and SEQ ID NO: 6 AQEAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKAILAEAKKLNWAQ

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

[0025] The properties of such linkers should preferably not destabilize the spatial three-dimensional structure of the protein units. This can be achieved, for example, by avoiding the presence of proline in the linker. Furthermore, the linker should preferably be sufficiently stable in an alkaline environment such that it does not impair the properties of the mutant protein units. 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 contain, at the N-terminus, a plurality of amino acid residues that constitute, for example, residues derived from a signal sequence that results from or is cleaved off during the cloning process. The number of additional amino acid residues can be 20 or less, for example 15 or less, for example 10 or less, or 5 or less. As a specific example, the multimer may contain AQ, AQGT, VDAKFDKE, AQVDAKFDKE, or AQGTVDAKFDKE (also referred to as a leader sequence) at the N-terminus. The multimer may contain, for example, a sequence as defined by SEQ ID NO: 7 or have at least 80%, at least 90%, or at least 95% identity thereto, or may consist essentially thereof. 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 polypeptide and / or multimer as disclosed above further comprises, at the C-terminus or N-terminus, one or more coupling elements selected from the group consisting of one or more cysteine residues, a plurality of lysine residues, and a plurality of histidine residues. The coupling element can also be located within 1 to 5 amino acid residues, such as 1 to 3 or 1 to 2 amino acid residues, from the C-terminus or N-terminus. The coupling element can be, for example, a single cysteine at the C-terminus. The coupling element can be directly linked to the C-terminus or N-terminus, or it / they can be linked via a stretch containing up to 15 amino acids, such as 1 to 5, 1 to 10, or 5 to 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 can be even more advantageous if the stretch does not contain glutamine. The advantage of having a C-terminal cysteine is that the end-point coupling of the protein can be achieved through the 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 ability.

[0027] The polypeptide or multimer can be attached to the support by conventional coupling techniques that utilize, for example, thiol groups (in cysteine), amino groups (in lysine or at the N-terminus), and / or carboxy 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. A molecule known as a spacer can be introduced between the support and the polypeptide / multimer, which improves the availability of the polypeptide / multimer and facilitates 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 multiple-point coupling (e.g., via multiple lysines) or 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 of performing such coupling are well known in the art and can be readily carried out by one of ordinary skill 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 through a terminal or near-terminal cysteine residue on the polypeptide or multimer, the mobility of the coupled polypeptide / multimer is enhanced, which provides an improvement in binding ability and binding reaction rate. In some embodiments, the polypeptide / multimer is coupled via the provided C-terminal cysteine on the protein as described above. This allows for efficient coupling of the cysteine thiol with an electrophilic group on the support, such as an epoxide group, a halohydrin group, etc., resulting in a thioether crosslink coupling.

[0029] In certain embodiments, the support comprises a polyhydroxy polymer such as a polysaccharide. Examples of polysaccharides include, for example, dextran, starch, cellulose, pullulan, agar, agarose, etc. Polysaccharides are essentially hydrophilic, have a low degree of non-specific interaction, they provide 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 reverse phase suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964)). Alternatively, the basic 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 specifically advantageous for large-scale separation, the support is adapted to increase its strong rigidity using the methods described in U.S. Patent No. 6,602,990 or No. 7,396,467 (incorporated herein by reference in their entirety), and thus, this makes the matrix more suitable for high flow rates.

[0031] In certain embodiments, a support such as a polymer, polysaccharide, or agarose support is cross-linked, such as by hydroxyalkyl ether cross-linking. Cross-linking reagents that cause such cross-linking can be epihalohydrins such as epichlorohydrin, diepoxides such as butanediol diglycidyl ether, or allylation reagents such as allyl halide or allyl glycidyl ether. Cross-linking is beneficial for the strong rigidity of the support and improves chemical stability. Hydroxyalkyl ether cross-linking is alkali-stable and does not cause significant non-specific adsorption.

[0032] In some embodiments, the solid support takes the form of a filter (e.g., a membrane or a depth filter matrix). Specifically, the support can comprise one or more sheets or membranes of cellulose nanofibers as described, for example, in U.S. Patent No. 9,802,979, No. 2016 / 0288089, and International Patent Application No. PCT / EP2019 / 050227, which are incorporated herein by reference in their entirety. The cellulose nanofibers may be appropriately cross-linked to improve chemical and mechanical stability.

Examples

[0033] (Example 1) Expression of a single-chain variable domain (V H H) A single-chain variable domain (V H H) derived from a camelid origin, also known as a nanobody, was heterologously expressed in Escherichia coli (E. coli). The V H H fragment was based on the sequence from K R Schmitz et al., Structure 21, 1214-1224, (2013) SEQ ID NO: 23: >4KRN:A|PDBID|CHAIN|SEQUENCE QVQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYTYYTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSLEHHHHHH and was modified for periplasmic expression by the use of the OmpA signal peptide (E. coli, outer membrane protein A, UniProt P0A910), with the signal peptide cleavage site introduced as the dipeptide AQ followed by an enzyme restriction site for KpnI (amino acids VD). The final protein after signal peptide processing had the sequence found in SEQ ID NO: 24 below:

[0034] AQVDQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYTYYTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSLEHHHHHH AQVDQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYTYYTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSLEHHHHHH

[0035] This array was generated by DNA synthesis company ATUM (CA, USA) using codon optimization from all 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 an ice bath and cooled for 20 minutes. Subsequently, a heat shock was applied at 42 °C for 60 seconds, followed by cooling on an ice bath for 2 minutes. After cooling the cells on ice, 950 μl of Luria broth (LB) was added and the cells were incubated with shaking at 225 rpm for 60 minutes. After incubation, the cells were plated on Luria agar (LA) plates at 100 μl per plate and the plates were incubated overnight in an oven set at 37 °C. The next day, single colonies were picked, grown in LB to a final OD 600nm of 1, then 15% glycerol was added and frozen at -80 °C until further use. The frozen cell bank was thawed and 100 μl was used to inoculate 100 mL of Terrific broth (TB) supplemented with 50 mg / L kanamycin and grown with shaking at 37 °C overnight. The next day, 10 mL of the overnight culture was used to inoculate 750 mL of fermentation medium supplemented with 50 mg / L kanamycin. The culture was induced with a final concentration of 1 mM IPTG when it reached an optical density (OD) measured at 600 nm of 80. The culture was maintained at a constant temperature and pH for 12 hours under aeration and agitation after induction. After 24 hours, fermentation was terminated and the cells were separated from the supernatant by centrifugation at 2500 x g for 30 minutes. The supernatant was discarded and the cells were resuspended in 600 mL of PBS and heat incubated at 48 °C for 3 hours to release the periplasmic expressed protein. The heat-treated suspension was centrifuged at 10000 x 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 of 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 and subsequently eluted with 50 mM citrate, pH 2.5. The 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) VH3 binding of V H H antibody fragment in different chromatography resins The VHH fragment prepared in Example 1 was 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 the hexameric ligand of SEQ ID NO: 7 by the method described in US Patent Application Publication No. 20180094024, which is hereby incorporated by reference in its entirety). All experiments were carried out 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 a column volume (CV) of 1.94 mL. All column chromatography runs were carried out using 20 mM phosphate, 500 mM NaCl, pH 7 as the running buffer, the second was a buffer consisting of 50 mM citrate, pH 6, and 50 mM citrate, pH 2.5 as the elution buffer. To test the binding to different resins, 10 mg of the V H H fragment prepared in Example 1 was diluted in 7 mL of running buffer (20 mM phosphate, pH 7, 500 mM NaCl) and loaded onto a 1 mL Mabselect™ Xtra, SuRe, or Prototype 1 column. The results from the chromatography runs are shown in FIGS. 2a)-c). The V H H fragment bound to the MabSelect™ Xtra column had a recovery rate of 73%, and the V HThe H fragment had a recovery rate of 86%. However, the fragment bound very weakly to MabSelect™ SuRe resin, with a recovery rate of only 4%, and most of the protein was found either in the loading step or the washing step. Furthermore, the dynamic binding capacity for Prototype 1 was determined for V H For the H fragment, it was measured by loading until 10% breakthrough was reached using a residence time of 6 minutes. Prototype 1 was packed into a Tricorn 5 / 100 column up to 1.98 mL CV. The measurement of its capacity was carried out using 20 mM phosphate, 150 mM NaCl, pH 7.4 as the running buffer, and 50 mM acetate, pH 3.5 as the elution buffer B. The column was regenerated between runs using 0.5 M NaOH. V H The H fragment was diluted to 2.3 mg / mL in 50 mL of 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 flowing the sample through the bypass. Sample addition was carried out at a flow rate of 0.33 mL / min (6-minute residence time) until the absorbance at 280 nm reached 10% of the maximum absorbance value. The washing step was carried out at a flow rate of 1 mL / min for 10 CV, followed by elution with a uniform concentration of buffer B. CIP was carried out using 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 36 mg / mL at 10% breakthrough (Q B10 ) using a 6-minute residence time.

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

[0038] (Example 3) VH3 Binding of Fab The VH3 type Fab was prepared from the full-length monoclonal antibody (mAb) by cleavage using papain. The mAb solution (54 ml, 1836 mg) was adjusted to pH 7.5 by adding 2M 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 next day, 400 μl of antipain (papain inhibitor) was added to the digested mAb. The solution was applied to a HiScale 26 / 135 Capto L column (GE Healthcare, Uppsala, Sweden) to purify the cleaved Fab fragment. 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 Superdex™ 200 Increase (GE Healthcare, Uppsala, Sweden) to verify complete cleavage of the mAb. The kinetic binding capacity for prototype 1 was measured for the Fab by loading to 10% breakthrough using a dwell time of 6 minutes. The Tricorn 5 / 100 column was packed with prototype 1 up to 1.98 mL CV. The capacity measurement was carried out using 20 mM phosphate, 150 mM NaCl pH 7.4 as the running buffer and 50 mM acetate, pH 3.5 as the elution buffer B. The column was regenerated with 0.5M NaOH between runs. The Fab was diluted to 2.25 mg / mL in 100 mL of 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 flowing the sample through the bypass. The sample addition was carried out at a flow rate of 0.33 mL / min (dwell time of 6 minutes) until the absorbance at 280 nm reached 10% of the maximum absorbance value.The washing process was carried out at a flow rate of 1 mL / min for 10 CV, followed by elution at a uniform concentration with buffer B. CIP was carried out at a flow rate of 1 mL / min for 3 CV using 0.5 M NaOH. Peaks greater than 100 mAU were collected at the selected outlet. The dynamic binding capacity for the prototype 1 resin was 106 mg / mL at 10% breakthrough (QB10) using a 6-minute residence time.

[0039] (Example 4) Four point mutations in the single-chain variable domain (V H H) fragment sequence To test the robustness of the interaction between protein A and the single-chain variable domain (V H H) fragment, a series of point mutations were generated and subsequently tested for retention of affinity for MabSelect™ Xtra, SuRe, and prototype 1. V HThe H array was generated by DNA synthesis company ATUM (CA, USA) using codon optimization for all 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 with shaking at 225 rpm for 60 minutes. After incubation, the cells were plated on Luria agar (LA) plates at 100 μl per plate and the plates were incubated overnight in an oven set at 37 °C. The next day, single colonies were picked, grown in LB to a final OD 600nm of 1, then 15% glycerol was added and frozen at -80 °C until further use. The frozen cell bank was thawed and 500 μl was used to inoculate 100 mL of Terrific broth (TB) supplemented with 50 mg / L kanamycin. The culture was grown at 37 °C until the OD 600nm reached 1, then induced with IPTG at a final concentration of 1 mM. After induction, the culture was grown overnight at 37 °C and stopped the next morning by centrifugation at 2300 xg for 20 minutes. The pellet was resuspended in 25 mM Tris-HCl, 500 mM NaCl, pH 8.0, then sonicated (40% amplitude, 2 minutes, 5 seconds on / 3 seconds off), centrifuged at 10000 xg for 10 minutes and filtered through a 0.2 μm filter. Then, the sample was added to columns of MabSelect™ Xtra, Mabselect™ SuRe, and prototype 1 to verify binding. The flow-through fractions were collected until elution peaks were confirmed.If no peak was obtained, the flow-through fraction was passed through a Ni-Sepharose FF column (GE Healthcare, Uppsala, Sweden) using 25 mM Tris pH 8.0 as the running buffer and a 5 CV gradient of 50 mM Tris, 250 mM imidazole, pH 8.0 as the 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 discloses the invention, including the best mode, by way of example, and enables any person skilled in the art to practice the invention, including making and using any device or system and performing any incorporated method. 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 only minor differences from the literal language of the claims. All patents and patent applications mentioned in this text are hereby incorporated by reference in their entirety as if individually incorporated.

Claims

**Claim 1** A method for the separation of an antibody or 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, wherein the ligand comprises a polypeptide defined by SEQ ID NO: 1 AQX 1 AFYEILX 2 LPNLTEEQRX 3 AFIQSLKDDPSVSKAILAEAKKLNX 4 AQ Array Number 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) and the polypeptide is as follows: AQEAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKAILAEAKKLNDAQ SEQ ID NO: 2 AQKAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKAILAEAKKLNDAQ SEQ ID NO: 3 AQEAFYEILKLPNLTEEQRNAFIQSLKDDPSVSKAILAEAKKLNDAQ SEQ ID NO: 4 AQEAFYEILHLPNLTEEQRAAFIQSLKDDPSVSKAILAEAKKLNDAQ SEQ ID NO: 5 and AQEAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKAILAEAKKLNWAQ SEQ ID NO: 6 and the antibody or antibody fragment binds to the separation resin; and c) eluting the antibody or antibody fragment from the separation resin with an elution liquid and recovering the antibody or antibody fragment A method comprising the above steps. **Claim 2** The method according to claim 1, wherein the polypeptide is defined by a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5 and 6. **Claim 3** The method according to claim 1 or 2, wherein the polypeptide is defined by the sequence shown in SEQ ID NO:

4. **Claim 4** The method according to any one of claims 1 to 3, wherein the polypeptide is defined by a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4 and 6. **Claim 5** The method according to any one of claims 1 to 4, wherein the polypeptide is defined by a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4 and 5. **Claim 6** The method according to any one of claims 1 to 5, wherein the ligand comprises a multimer of the polypeptide linked by a linker region containing 0 to 15 amino acid residues. **Claim 7** The method according to claim 6, wherein the multimer is a tetramer, pentamer or hexamer. **Claim 8** The method according to 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. The method according to any one of claims 1 to 8, wherein the support comprises crosslinked agarose beads.

10. The method according to any one of claims 1 to 9, wherein the support comprises crosslinked cellulose nanofibers.

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

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

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 within the nanomolar range or stronger.

15. The method according to any one of claims 1 to 14, wherein the antibody or antibody fragment has the ability to bind to native protein A via the VH3 region.

16. The method according to any one of claims 1 to 15, wherein the method includes a step of washing the separation resin with a washing liquid, and contaminants and / or impurities are removed between steps b) and c).

17. The method according to claim 16, wherein the washing liquid is a buffer at pH 5 to 7.

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

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

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

21. The method according to claim 19 or 20, wherein steps a) to d) are repeated at least 50 times.

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