Method for isolating antibodies or antibody fragments that lack an Fc region capable of binding to protein A

Abstract

The present invention discloses a method for isolating an antibody or antibody fragment, comprising the following steps: 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) a step of contacting a feed with a separation resin having a covalently coupled ligand, wherein the ligand comprises a polypeptide as defined by sequence identification number: 1, and an antibody or antibody fragment binds to the separation resin; c) Optionally, a step of washing the separated resin with a washing solution; d) a step of eluting the antibody or antibody fragment from the separation resin into an eluent and recovering the antibody or antibody fragment.

Description

Technology Field

[0001] The present invention relates to a method for isolating immunoglobulins, more particularly to isolating an antibody or antibody fragment having a VH3 region and lacking 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 ligands selectively bind to the Fc region of immunoglobulins, enabling a highly efficient capture step. For antibody constructs lacking the Fc region, e.g., antibody fragments, or for immunoglobulins having Fc region variants that do not bind to Protein A, e.g., IgG3 or IgM, natural Protein A may still be useful in that it also binds to the VH3 region of the immunoglobulin. However, natural Protein A is not stable under the alkaline washing conditions used in bioprocessing, and alkali-stabilized Protein A variants generally contain mutations that inhibit VH3 interactions (e.g., see US20060194950 incorporated herein by reference). This document [refers to] the commercially available product MabSelect TM We discuss the inhibition of VH3 interactions by the G29A mutation in the Protein A Fc-binding B domain used in SuRe. Alternative ligands capable of binding to antibodies / fragments lacking the Fc region include Protein L, Protein G, and camel antibodies. However, these are all highly sensitive to alkaline washing conditions.

[0003] Therefore, there is a need for an affinity chromatography separation method for antibodies or antibody fragments that do not have an Fc region capable of binding to protein A, using a separation resin that withstands alkaline washing.

[0004] One aspect of the present invention is to provide a method for isolating an antibody or an antibody fragment. This is achieved by a method comprising the following steps:

[0005] a) a step of providing a feed comprising an antibody or antibody fragment having a VH3 region and lacking an Fc region that can bind to protein A;

[0006] b) a step of contacting a feed with a separation resin having a ligand covalently coupled to a support, wherein the ligand comprises a polypeptide as defined by the following SEQ ID NO: 1, and an antibody or antibody fragment is bound to the separation resin:

[0007]

[0008] In the above formula,

[0009] X1= E,K,Y,T,F,L,W,I,M,V,A,H or R,

[0010] X2 = H or K,

[0011] X3 = N or A,

[0012] X4= D, F, Y, W, K or R;

[0013] c) optionally, a step of washing the separated resin with a washing solution; and

[0014] d) a step of eluting the antibody or antibody fragment from the separation resin into an eluent and recovering the antibody or antibody fragment.

[0015] One advantage is that this method can separate antibodies without an Fc region capable of binding to protein A. An additional advantage is that the ligand and resin are alkali-stable and withstand repeated washing cycles with up to 2 M NaOH.

[0016] Further suitable embodiments of the present invention are described in dependent claims. Brief explanation of the drawing

[0017] Figure 1 shows the structure of a) a typical IgG antibody and b) a Fab fragment. Figure 2 shows the chromatogram of 10 mg VHH-EgA1 on a) MapSelect Extra (Xtra); b) MapSelect Cart; and c) Prototype 1. Figure 3 shows the chromatogram of 30 mg / mL VHH on prototype 1. Specific details for implementing the invention

[0018] definition

[0019] The terms "antibody" and "immunoglobulin" are used interchangeably herein and are understood to include fragments of antibodies, fusion proteins comprising antibodies or antibody fragments, and conjugates comprising antibodies or antibody fragments.

[0020] The terms "Fc-binding polypeptide" and "Fc-binding protein" each refer to a polypeptide or protein 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 that retains said binding properties.

[0021] In this document, the term "antibody or antibody fragment lacking an Fc region capable of binding to protein A" means 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, and camel V HIt includes H single-domain antibodies and any antibody having an Fc region engineered to not bind to protein A. An actual test to determine whether a specific antibody / fragment has an Fc region capable of binding to protein A is performed using MAPSelect, a non-VH3-binding protein A resin, in 20 mM phosphate, 500 mM NaCl, pH 7 loading buffer. TM The procedure involves loading the column packed with a cart (GE Healthcare), washing the column first with loading buffer, then washing with 50 mM citrate pH 6 wash buffer, and finally eluting the column with 50 mM citrate pH 2.5 elution buffer. If the antibody / fragment does not possess an Fc region capable of binding to protein A, it will be found primarily in the loading pass-through and / or wash buffer. If it possesses such an Fc region, it will be found primarily in the eluent.

[0022] In this document, the term "linker" means an element that links two polypeptide units, monomers, or domains together in a multimer.

[0023] In this document, the term "spacer" refers to an element that connects a polypeptide or polypeptide polymer to a support.

[0024] The term "identity %" for the comparison of amino acid sequences is, for example, the basic local alignment tool (BLAST) described in the literature (Altshul et al. (1990) J. Mol. Biol., 215: 403-410) TM It is determined by standard alignment algorithms such as ). Web-based software for this is from the U.S. National Library of Medicine http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome It is freely available at. Here, the algorithm "blastp (protein-protein blast)" is used to align the query sequence and the target sequence and to determine the ia identity percentage.

[0025] In this document, the term "natural protein A" means a polypeptide comprising five naturally occurring immunoglobulin-binding domains E, D, A, B, and C that are directly linked to each other, as defined by sequence identification number: 31. The polypeptide may further comprise a leader sequence at the N-terminal end and a coupling element at the C-terminal or N-terminal end.

[0026] Terms used herein such as “include,” “comprising,” “containing,” “having,” etc., may have the meanings given to them under U.S. patent law and may mean “include,” “comprising,” etc.; “essentially composed of” or “essentially composed of,” etc., have the meanings given to them under U.S. patent law, and the terms exclude open or prior art embodiments that allow for more than the cited, unless the basic or novel features of the cited are altered by the existence of more than the cited.

[0027] Detailed description of the embodiment

[0028] In one aspect, the present invention relates to Staphilococcus ( Staphylococcus A method for isolating an antibody or antibody fragment that does not have an Fc region capable of binding to protein A is disclosed. The method comprises the following steps:

[0029] a) a step of providing a feed, such as purified cell culture supernatant, comprising an antibody or antibody fragment having a VH3 region and lacking an Fc region that can bind to protein A. In particular, the target species in the feed to be isolated may be an antibody fragment having a VH3 region and lacking an Fc region. These may be selected from the group consisting of, for example, Fab, scFv, domain antibodies, nanobodies, and BiTe: The target species in the feed to be isolated may also be an antibody having a VH3 region and an Fc region that does not bind to protein A. These may be, for example, IgG3, IgM, and camel V HIt may be selected from the group consisting of H single-domain antibodies. Overall, the antibody or antibody fragment can bind to natural protein A through the VH3 region. For non-Fc antibodies / fragments, a MAP-Select comprising a covalently coupled natural protein A ligand according to the above definition TM Alternatively, this can be easily tested by checking whether it binds to MAPSelect Extra Resin (GE Healthcare).

[0030] b) a step of contacting a feed with a separation resin having a ligand covalently coupled to a support, wherein the ligand comprises a polypeptide defined by sequence identification number: 1 or having at least 85%, e.g., at least 90% or at least 95% identity therewith, and an antibody or antibody fragment is bound to the separation resin:

[0031]

[0032] In the above formula,

[0033] X1= E,K,Y,T,F,L,W,I,M,V,A,H or R,

[0034] X2 = H or K,

[0035] X3 = N or A,

[0036] X4= D, F, Y, W, K or R.

[0037] The binding strength of antibodies or antibody fragments to the separation resin can be in the nanomolar range or stronger.

[0038] c) Optionally, a step of washing the separation resin with a washing solution to remove contaminants and / or impurities. The washing solution may be, for example, a buffer with a pH of 5-7.

[0039] d) a step of eluting the antibody or antibody fragment from the separation resin into an eluent and recovering the antibody or antibody fragment. The eluent may suitably be a buffer with a pH of 2-5.

[0040] After step d), the method may further include step e) of washing the separation resin with a washing solution having a pH of 13 or higher. The washing solution may include an alkali metal hydroxide, such as NaOH, with a pH of 0.1 to 2 M, for example, 0.5 to 2 M.

[0041] In some embodiments, steps a) - e) are repeated at least 50 times, e.g., at least 200 times.

[0042] In certain embodiments, X1= E, X2= H, X3= N and / or X4= D. Examples of such embodiments include the following:

[0043]

[0044] In some embodiments, the ligand comprises a multimer of polypeptide P. This multimer can be suitably linked by a linker region L comprising 0 to 25 amino acid residues, e.g., 0 to 15 amino acid residues, e.g., structure (P - L) n-1 - P or L-(P - L) n-1- It may have P. The polymer may be, for example, a dimer, trimer, tetramer, pentamer, hexamer, heptmer, octamer, or gypsum. All units of the polymer may be homomers, or at least one unit may be a heteromer, different from the other units. Advantageously, all units of the polymer are stable to alkali by including, for example, the mutation disclosed above. The polypeptides may be directly connected to each other by peptide bonds between the C-terminal and N-terminal ends of the polypeptides. Alternatively, two or more units of the polymer may be connected by a linker comprising an oligomer or polymer species, such as a linker comprising 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 may comprise or essentially consist of a peptide sequence defined by an amino acid sequence selected from the group consisting of, for example, 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, or having at least 80%, at least 90%, or at least 95% identity with such a sequence. These may also essentially consist of peptide sequences defined by amino acid sequences selected from the group consisting of APKADNKFNKE, APKVFDKE, APAKFDKE, AKFDKE, APKVDA, VDAKFDKE, APKKFDKE, APK and APKYEDGVDAKFDKE, or having at least 80%, at least 90%, or at least 95% identity with this.

[0045] The characteristics of such a linker should preferably not destabilize the spatial stereostructure of the protein unit. This can be achieved, for example, by avoiding the presence of proline in the linker. Additionally, the linker should preferably be sufficiently stable in an alkaline environment so as not to impair the characteristics of the mutated protein unit. To this end, it is advantageous for the linker not to contain asparagine. It may be additionally advantageous if the linker does not contain glutamine. The multimer may additionally include a plurality of amino acid residues at the N-terminus that constitute residues from the cloning process or from a cleaved signaling sequence. The number of additional amino acid residues may be, for example, 20 or fewer, for example 15 or fewer, for example 10 or fewer, or 5 or fewer. As a specific example, the multimer may include the sequence AQ, AQGT, VDAKFDKE, AQVDAKFDKE, or AQGTVDAKFDKE (also referred to as a leader sequence) at the N-terminus. The multimer may include or essentially consist of a sequence defined by, for example, sequence identification number: 7, or having at least 80%, at least 90%, or at least 95% identity with it.

[0046]

[0047] In some embodiments, the polypeptide and / or multimer disclosed above further comprises 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 at the C-terminal or N-terminal end. The coupling element(s) may also be located within 1 to 5 amino acid residues from the C-terminal or N-terminal end, e.g., within 1 to 3 or 1 to 2 amino acid residues. The coupling element may be, e.g., a single cysteine ​​at the C-terminal end. The coupling element(s) may be directly connected to the C- or N-terminal end, or they may be connected via a stretch comprising up to 15 amino acids, e.g., 1 to 5, 1 to 10, or 5 to 10 amino acids. This stretch should preferably be sufficiently stable in an alkaline environment so as not to impair the properties of the mutated protein. To this end, it is advantageous for the stretch not to contain asparagine. It may be further advantageous if the stretch does not contain glutamine. The advantage of having a C-terminal cysteine ​​is that endpoint coupling of the protein can be achieved through the reaction of cysteine ​​thiol with electrophilic groups on the support. This provides excellent mobility of the coupled protein, which is important for binding capacity.

[0048] Polypeptides or polymers can be attached to a support via conventional coupling techniques utilizing thiol (in cysteine), amino (in lysine or the N-terminus), and / or carboxyl (in aspartic acid or glutamic acid or the C-terminus) groups present in the ligand, for example. Bisecoxide, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS), etc., are widely Golgi-established coupling agents. Molecules known as spacers may be introduced between the support and the polypeptide / multimer, which improve the utility of the polypeptide / multimer and facilitate the chemical coupling of the polypeptide / multimer to the support. Depending on the characteristics of the polypeptide / multimer and the coupling conditions, the coupling may be multi-point coupling (e.g., through multiple lysines) or single-point coupling (e.g., through a single cysteine).

[0049] In certain embodiments, a polypeptide or multimer is coupled to a support via a thioether bond. Methods for performing such coupling are widely known in the art and can be easily performed by a person skilled in the art using standard techniques and equipment. Thioether bonds are flexible, stable, and generally suitable for use in affinity chromatography. In particular, when the thioether bond is formed through terminal or near-terminal cysteine ​​residues on the polypeptide or multimer, the mobility of the coupled polypeptide / multimer is enhanced, providing improved binding capacity and binding kinetics. In some embodiments, the polypeptide / multimer is coupled via C-terminal cysteine ​​provided on the protein as described above. This efficiently couples the cysteine ​​thiol to electrophilic groups on the support, such as epoxide groups, halohydrin groups, etc., to create a thioether bridge coupling.

[0050] In certain embodiments, the support comprises a polyhydroxy polymer such as a polysaccharide. Examples of polysaccharides include, for instance, dextran, starch, cellulose, pullulan, agar, agarose, etc. Polysaccharides are inherently hydrophilic with a low degree of non-specific interaction, provide a high content of reactive (activable) hydroxyl groups, and are generally stable to alkaline washing solutions used in bioprocessing.

[0051] In some embodiments, the support comprises agar or agarose. The support used in the present invention can be readily prepared according to standard methods such as reverse suspension gelation (S. Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964)). Alternatively, the base matrix may be a commercially available product, such as Sepharose TM It is a cross-linked agarose bead sold under the name FF (GE Healthcare). In one embodiment particularly advantageous for large-scale separation, the support is adjusted to increase stiffness using the method described in US6602990 or US7396467 (the full text incorporated herein by reference), thus making the matrix more suitable for high flow rates.

[0052] In certain embodiments, a support such as a polymer, polysaccharide, or agarose support is crosslinked, such as by hydroxyalkyl ether crosslinking. The crosslinking agent reagent that produces this crosslinking may be an epihalohydrin, such as epichlorohydrin; a diepoxide, such as butanediol diglycidyl ether; or an allylation agent, such as an allyl halide or allyl glycidyl ether. Crosslinking is beneficial to the stiffness of the support and improves its chemical stability. Hydroxyalkyl ether crosslinking is stable to alkali and does not cause significant nonspecific adsorption.

[0053] In some embodiments, the solid support is in the form of a filter (e.g., a membrane or a deep filter matrix). In particular, the support may comprise one or more sheets or membranes of cellulose nanofibers, as described, for example, in US9802979, US20160288089 and PCT / EP2019 / 050227 (the full text of which is incorporated herein by reference). The cellulose nanofibers may be suitably crosslinked to improve chemical and mechanical stability.

[0054] Examples

[0055] Example 1: Single heavy chain variable domain (V H Expression of H)

[0056] Camellia, also announced as a nanobody ( Camelidae Single heavy chain variable domain (V) from ) origin H H) This Esherikia Kolai ( Escherichia coli ) (i. colai ( E.coli It was expressed as a heterogeneous form in )). V H The H fragment was based on the sequence from the literature (KR Schmitz et al:, Structure 21, 1214-1224, (2013)), sequence identification number: 23 below:

[0057] >4KRN:A|PDBID|CHAIN|SEQUENCE

[0058]

[0059] This sequence was modified for peripheral cytoplasmic expression using the OmpA signal peptide (E. coli, outer membrane protein A, UniProt P0A910), and after the signal peptide cleavage site was introduced as dipeptide AQ, an enzymatic restriction site for KpnI (amino acid VD) was introduced. The final protein after signal peptide processing had the sequence found at the following sequence identification number: 24:

[0060]

[0061] This sequence was generated by the DNA synthesis company ATUM (California, USA) using proprietary codon optimization from E. coli expression and cloned into an expression vector (plasmid) containing an IPTG-induced promoter. The plasmid was transformed into chemically qualified 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 qualified cells in an ice bath and kept cold for 20 minutes. Heat shock was applied at 42°C for 60 seconds, followed by cooling in 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 in a shake hood at 225 rpm for 60 minutes. After incubation, 100 μl of cells were plated onto Luria agar (LA) plates and incubated overnight in an oven set to 37°C. The next day, a single colony was selected and grown in LB until the final OD at 600 nm was 1, then 15% glycerol was added, and the cells were frozen at -80°C for further use. The frozen cell bank was thawed, 100 μl was used to inoculate 100 mL of terrific broth (TB) supplemented with 50 mg / L kanamycin, and the cells were grown overnight at 37°C in a shake hood. The next day, 10 mL of the overnight culture was used to inoculate 750 mL of fermentation medium supplemented with 50 mg / L kanamycin. When the optical density (OD) measured at 600 nm reached 80, the culture was induced with a final concentration of 1 mM IPTG. After induction, the culture was maintained at a constant temperature and pH for 12 hours under aeration and stirring. Fermentation was terminated after 24 hours, and the cells were separated from the supernatant by centrifugation at 2500 xg for 30 minutes.The supernatant was poured off, cells were resuspended in 600 mL of PBS, and heat-incubated at 48°C for 3 hours to release peripheral cytoplasmic expressed proteins. The heat-treated suspension was centrifuged at 10,000 xg for 30 minutes, followed by sterile filtration using a 0.2 μm filter. The purified supernatant was saturated with 24 mL of MAPSelect equilibrated with 20 mM phosphate, pH 7, and 500 mM NaCl. TM It was loaded onto a HiScale 16 column (GE Healthcare, Uppsala, Sweden) packed with resin (GE Healthcare, Uppsala, Sweden). The column was washed with 50 mM citrate pH 6 to reduce the conductivity of the elution pool, and then eluted 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 adding 2 M Tris base.

[0062] Example 2: V on different chromatographic resins H VH3 binding of H antibody fragment

[0063] The VHH fragment generated in Example 1 is treated with a different chromatography resin, MapSelect TM Extra (GE Healthcare, Uppsala, Sweden), MAPSelect TM Binding was tested for highly cross-linked agarose beads with a central bead diameter (d50,v) of 57 μm coupled with the hexameric ligand of SEQ ID No. 7 according to the methods described in Sure (GE Healthcare, Uppsala, Sweden) and Prototype 1: US20180094024 (the full text incorporated herein by reference). All experiments were performed using an AKTA Pure 150 FPLC system (GE Healthcare, Uppsala, Sweden). MAPSelect TM Extras and Map Select TM The cart is a pre-packed 1 mL HiTrap TMTests were performed using a column (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 chromatographic runs were performed using a second buffer consisting of 20 mM phosphate, 500 mM NaCl, pH 7, and 50 mM citrate pH 6 as the run buffer, and 50 mM citrate pH 2.5 as the elution buffer. To test binding to different resins, 10 mg V produced in Example 1 H Dilute the H fragment in 7 mL of running buffer (20 mM phosphate pH 7, 500 mM NaCl), and 1 mL of MAPSelect TM It was loaded onto Extra, Cart, or Prototype 1 columns. The results of the chromatography runs are shown in Figures 2a)-c). V H H short story MapSelect with a 73% recovery rate TM It was combined to the extra column and combined to Prototype 1 with an 86% recovery rate. However, the fragment was MapSelect with only a 4% recovery rate. TM It bound very weakly to the cart resin, and most proteins were detected during the loading or washing phases. Additionally, the dynamic binding capacity for Prototype 1 was measured by loading until 10% breakthrough was reached using a 6-minute retention time, V H Measurements were performed on the H fragment. A Tricon 5 / 100 column was packed with Prototype 1 in a 1.98 mL CV. Volume measurements were performed using elution buffer B containing 20 mM phosphate, 150 mM NaCl pH 7.4, and 50 mM acetate pH 3.5 as the run buffer. The column was regenerated between runs using 0.5 M NaOH. V HThe H fragment was diluted to 2.3 mg / mL in 50 mL of run 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 bypass. Sample application was performed at a flow rate of 0.33 mL / min (6-minute retention time) until the absorbance at 280 nm reached 10% of the maximum absorbance. A wash step was performed at a flow rate of 1 mL / min for 10 CVs, followed by isolytic elution with Buffer B. CIP was performed with 0.5 M NaOH at a flow rate of 1 mL / min for 3 CVs. Peaks greater than 100 mAU were collected at selected outlets. The dynamic binding capacity for Prototype 1 resin was determined at 10% breakthrough (Q) using a 6-minute retention time. B10 It was 36 mg / mL.

[0064] Fig. 2a)

[0065] Column: MapSelect TM extras

[0066] Sample: 10 mg VHH-EgA1 (Sequence Identification Number: 25)

[0067] Buffer: Starting: 20 mM phosphate, 500 mM NaCl, pH 7

[0068] Wash 2: 50 mM citrate pH 6

[0069] Elution: 50 mM citrate pH 2.5

[0070] Fig. 2b)

[0071] Column: MapSelect TM wagon

[0072] Sample: 10 mg VHH-EgA1 (Sequence Identification Number: 25)

[0073] Buffer: Starting: 20 mM phosphate, 500 mM NaCl, pH 7

[0074] Wash 2: 50 mM citrate pH 6

[0075] Elution: 50 mM citrate pH 2.5

[0076] Fig. 2c)

[0077] Column: Prototype 1

[0078] Sample: 10 mg VHH-EgA1 (Sequence Identification Number: 25)

[0079] Buffer: Starting: 20 mM phosphate, 500 mM NaCl, pH 7

[0080] Wash 2: 50 mM citrate pH 6

[0081] Elution: 50 mM citrate pH 2.5

[0082] Example 3: VH3 binding of Fab

[0083] VH3 type Fab was generated from a full-length monoclonal antibody (mAb) by cleavage using papain. An mAb solution (54 ml, 1836 mg) was adjusted to pH 7.5 by adding 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 (21 mg) were added to the solution. The solution was incubated overnight at 37°C. The next day, 400 μl of Antipain (a papain inhibitor) was added to the digested mAb. The solution was fed into a Highscale 26 / 135 Capto L column (GE Healthcare, Uppsala, Sweden) to purify the cleaved Fab fragment. The decomposed 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. Superdex was used to confirm the complete cleavage of the mAb. TMAnalysis was performed by SEC using a 200 Increase (GE Healthcare, Uppsala, Sweden). Dynamic binding capacity for Prototype 1 was measured for Fab by loading until 10% breakthrough was reached using a retention time of 6 minutes. A Tricon 5 / 100 column was packed with Prototype 1 in a 1.98 mL CV. Capacity measurements were performed using elution buffer B containing 20 mM phosphate, 150 mM NaCl pH 7.4, and 50 mM acetate pH 3.5 as the run buffer. The column was regenerated between runs using 0.5 M NaOH. Fab was diluted to 2.25 mg / mL in 100 mL of run 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 bypass. Sample application was performed at a flow rate of 0.33 mL / min (6-minute retention time) until the absorbance at 280 nm reached 10% of the maximum absorbance. A washing step was performed at a flow rate of 1 mL / min for 10 CVs, followed by isolytic elution with Buffer B. CIP was performed with 0.5 M NaOH at a flow rate of 1 mL / min for 3 CVs. Peaks greater than 100 mAU were collected at selected outlets. The dynamic binding capacity for Prototype 1 resin was 106 mg / mL at 10% breakthrough (QB10) at a 6-minute retention time.

[0084] Example 4: Single heavy chain variable domain (V H H) Point mutation of fragment sequence

[0085] Protein A and single heavy chain variable domain (V H H) To test the robustness of interactions between fragments, create a set of point mutants, then MapSelect TM Retention affinity for Extra, Cart, and Prototype 1 was tested. V HThe H sequence was generated by DNA synthesis company ATUM (California, USA) using proprietary codon optimization for E. coli expression and cloned into an expression vector (plasmid) containing an IPTG-induced promoter. The plasmid was transformed into chemically qualified 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 qualified cells in an ice bath and kept cold for 20 minutes. Subsequently, a heat shock was applied at 42°C for 60 seconds, followed by cooling in 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 in a shake hood at 225 rpm for 60 minutes. After incubation, 100 μl of cells were plated onto Luria agar (LA) plates and incubated overnight in an oven set to 37°C. The next day, a single colony was selected and grown in LB until a final OD 600 nm of 1 was reached; then, 15% glycerol was added, and the cells were frozen at -80°C for further use. The frozen cell bank was thawed, and 500 μl was used to inoculate 100 mL of terepic broth (TB) supplemented with 50 mg / L kanamycin. The culture was grown at 37°C until an OD 600 nm of 1 was reached, followed by induction with a final concentration of 1 mM IPTG. After induction, the culture was grown overnight at 37°C and centrifuged at 2300 xg for 20 minutes to discontinue the following morning. The pellet was resuspended in 25 mM Tris-HCl, 500 mM NaCl, pH 8.0, then sonicated (40% amplitude, 2 min, 5 sec on / 3 sec off), centrifuged at 10,000 xg for 10 minutes, and filtered through a 0.2 μm filter. Subsequently, the sample was MAPSelect TM Extra, Map Select TMBinding was confirmed by applying to the Cart and Prototype 1 columns. The pass fraction was collected until the elution peak was confirmed. If no peak was obtained, the Ni-Sepharose FF column (GE Healthcare, Uppsala, Sweden) was run on the pass fraction using a 5 CV gradient elution with 25 mM Tris pH 8.0 and 50 mM Tris, 250 mM imidazole pH 8.0 as run buffers.

[0086]

[0087] Y = Confirmed bonds present, N = Confirmed bonds absent.

[0088]

[0089]

[0090] The description in this specification discloses the invention, including in the best mode, and uses examples to enable a person skilled in the art to practice the invention, including making and using any device or system and performing any included method. The patentable scope of the invention is defined by the claims and may include other examples that occur to a person skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that are not different from the literal language of the claims or include equivalent structural elements that are substantially different from the literal language of the claims. All patents and patent applications mentioned in the text are incorporated by reference in their entirety, as if individually included.

Claims

Claim 1 A method for separating an antibody or antibody fragment, comprising the following steps: 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 as defined by sequence identification number 2, and the antibody or antibody fragment binds to the separation resin; and d) eluting the antibody or antibody fragment from the separation resin into an eluent and recovering the antibody or antibody fragment. Claim 2 A method according to claim 1, wherein the ligand comprises a multimer of the polypeptide connected by a linker region containing 0 to 15 amino acid residues. Claim 3 In paragraph 2, the method wherein the above-mentioned macromer is a tetramer, pentamer, or hexamer. Claim 4 A method according to claim 2 or 3, wherein the multimer is coupled to the support through a thioether link derived from a C-terminal cysteine ​​on the multimer. Claim 5 A method according to any one of claims 1 to 3, wherein the support comprises cross-linked agarose beads. Claim 6 A method according to any one of claims 1 to 3, wherein the support comprises cross-linked cellulose nanofibers. Claim 7 A method according to any one of claims 1 to 3, wherein the feed is a purified cell culture supernatant. Claim 8 A method according to any one of claims 1 to 3, wherein the antibody or antibody fragment is an antibody fragment having a VH3 region and not an Fc region. Claim 9 A method according to any one of claims 1 to 3, wherein the antibody fragment is selected from the group consisting of Fab, scFv, domain antibodies, nanobodies, and BiTe. Claim 10 A method according to any one of claims 1 to 3, wherein the binding strength of the antibody fragment to the separation resin is in the nanomolar range or stronger. Claim 11 A method according to any one of claims 1 to 3, wherein the antibody or antibody fragment is capable of binding to natural protein A through the VH3 region. Claim 12 A method according to any one of claims 1 to 3, wherein the method further comprises, prior to step d), step c) of washing the separation resin with a washing solution, and wherein contaminants and / or impurities are removed in step c). Claim 13 In claim 12, the method wherein the washing solution is a buffer with a pH of 5-7. Claim 14 A method according to any one of claims 1 to 3, wherein the eluent is a buffer with a pH of 2-5. Claim 15 A method according to any one of claims 1 to 3, further comprising, after step d), step e) of washing the separation resin with a washing solution having a pH of 13 or higher. Claim 16 A method according to claim 15, wherein the cleaning solution comprises 0.5 - 2 M of alkali metal hydroxide. Claim 17 A method according to claim 15, wherein steps a), b), d), and e) are repeated at least 50 times. Claim 18 A method according to any one of claims 1 to 3, wherein the method further comprises, prior to step d), step c) of washing the separation resin with a washing solution. Claim 19 A method according to claim 18, further comprising step e) of washing the separation resin with a washing solution having a pH of 13 or higher after step d). Claim 20 In paragraph 19, a method in which steps a) - e) are repeated at least 50 times. Claim 21 delete

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